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Novex® Pre-Cast Gel Electrophoresis Guide

1. 13 14 15 Place one side of the DryEase Gel Drying Frame with the corner pin facing up on the DryEase Gel Drying Base Center a piece of pre wetted cellophane from Step 5 over the base frame combination so the cellophane lays over the inner edge of the frame Lay the gel on the center of the cellophane sheet making sure no bubbles are trapped between the gel and the cellophane Add some Gel Dry Drying Solution to the surface of the cellophane if necessary Carefully lay the second sheet of cellophane over the gel so that no bubbles are trapped between the cellophane and the gel Add some Gel Dry Drying Solution if necessary Gently smooth out any wrinkles in the assembly with a gloved hand Align the remaining frame so that its corner pins fit into the appropriate holes on the bottom frame Push the plastic clamps onto the four edges of the frames Lift the frame assembly from the DryEase Gel Drying Base and pour off the excess solution from the base Stand the gel dryer assembly upright on a bench top Be careful to avoid drafts as they can cause an uneven rate of dying which leads to cracking Drying takes between 12 36 hours depending on humidity and gel thickness When the cellophane is dry to touch remove the gel cellophane sandwich from the drying frame Trim off the excess cellophane Press the dried gel s between the pages of a notebook under light pressure for approximately 2 days so they remain
2. Std Strip e For 1 5 mm Thick Gels Wet two pieces of thin filter paper 5 8 x 4 cm in 1X SDS Running Buffer Sandwich and the IEF gel strip with the filter paper such that the edge of the gel strip protrudes 0 5 mm beyond the edge of the paper see figure below Insert the sandwich into the well and push the strip so it comes in contact with the gel Avoid trapping air bubbles between the gel strip and the surface of the gel Filter Paper Filt y Paper LAO Ta TT IEF Gel Strip Electrophoresis See page 32 for instructions on running Novex Pre Cast Gels using the XCell Conditions SureLock Mini Cell Run the gel at 125 V constant After the dye front has moved into the stacking gel 10 min disconnect the power supply remove the filter paper and resume electrophoresis to completion Staining the Gel Stain the gel with the appropriate method for the type of gel and sample amount after electrophoresis Refer to the techniques described on pages 35 46 23 ZOOM Gels ZOOM Gels 2D Separation of IPG Strips Materials Supplied by the User Equilibrating the IPG Strip 24 ZOOM Gels are used for 2D analysis of proteins following isoelectric focusing of IPG strips ZOOM Gels are 1 0 mm thick and contain an IPG well and a molecular weight marker well The IPG well is designed to accommodate a 7 0 cm IPG strip Two types of ZOOM Gels are available see page 63 for ordering information
3. 4 Tris Glycine Gels Tricine Gels Zymogram Gels IEF Gels TBE Gels TBE Urea Gels DNA Retardation Gels Important Formulation Stacking Gel Separating Gel Bis pH Acrylamide Tris base HCl 4 6 8 10 12 2 6 8 6 Acrylamide Bis 14 16 18 acrylamide TEMED 4 12 8 16 APS Ultrapure water 4 20 10 20 Tris base HCl 4 10 16 10 20 2 6 8 3 Acrylamide Bis acrylamide TEMED APS Ultrapure water Tris Glycine Gels with 4 10 12 4 16 2 6 8 6 a substrate casein or No substrate gelatin Acrylamide Bis None pH 3 7 2 6 5 0 acrylamide TEMED pH 3 10 6 0 APS Ultrapure water 2 ampholytes Tris base Boric acid 4 6 8 10 20 2 6 8 3 EDTA Acrylamide 4 12 4 20 Bis acrylamide TEMED APS Ultrapure water Tris base Boric acid 4 6 10 15 3 8 5 8 7 EDTA Acrylamide Bis acrylamide TEMED APS Ultrapure water 7M Urea 6 polyacrylamide None 6 2 6 8 3 gels prepared with half strength TBE gel buffer Novex Pre Cast gels do not contain SDS These gels can be used for non denaturing native and denaturing gel electrophoresis For optimal and total separation ranges for each specific gel percentage consult the Gel Migration Charts on page 72 Gel Selection Choosing a Gel for Your Application Protein Separation Applications To obtain the best results it is important to choose the correct gel percentage b
4. Tricine SDS Running Buffer 10X Novex Tricine SDS Sample Buffer 2X Novex Zymogram Renaturing Buffer 10X Novex Zymogram Developing Buffer 10X Novex TBE Running Buffer 5X Novex Hi Density TBE Sample Buffer 5X Novex TBE Urea Sample Buffer 2X Novex Prep TBE Urea Sample Buffer 2X NuPAGE Novex 4 12 Bis Tris ZOOM Gel Novex 4 20 Tris Glycine ZOOM Gel UltraPure Agarose Nitrocellulose 0 45um Invitrolon PVDF 0 45 um Nylon 0 45 um Novex pH 3 7 IEF Buffer Kit includes LC5300 LC5370 LC5371 Novex pH 3 10 IEF Buffer Kit includes LC5300 LC5310 LC5311 Quantity Catalog no 1 unit E10001 1 unit EI9051 1 unit E18600 1 kit NI2387 2 pack NI2400 500 mL LC4025 1 unit 1B1001 1 unit SD1000 500 mL LC2675 250 pL NP0004 250 mL NP0008 500 mL LC3675 500 mL LC2672 20 mL LC2673 20 mL LC2676 500 mL LC1675 20 mL LC1676 500 mL LC2670 500 mL LC2671 LC6675 10 mL LC6678 10 mL LC6876 20 mL LC6877 1 gel NP0330BOX 1 gel EC60261BOX 1 kit LC5377 1 kit LC5317 100 g 15510 019 20 membrane filter papers LC2000 20 membrane filter papers LC2005 m 20 membrane filter papers LC2003 Continued on next page 63 Accessory Products Continued Protein Stains and Ordering information for stains and protein molecular weight standards is Standards provided below For more information visit our website at www invitrogen com or contact Techn
5. 4 Prepare Reducing Solution by diluting 250 uL of the NuPAGE Sample Reducing Agent 10X in 1 75 mL of 1X SDS Sample Buffer 5 Incubate the strip in Reducing Solution for 3 5 minutes Decant the Reducing Solution 6 Prepare 125 mM Alkylating Solution by adding 58 mg of fresh iodoacetamide to 2 5 mL of 1X SDS Sample Buffer Incubate the strip in Reducing Solution for 3 5 minutes 8 Decant the Alkylating Solution and proceed to 2D Separation of Proteins on Novex IEF Gels next page IEF Gels Continued 2D Separation of A protocol for separating proteins in an IEF gel strip by SDS PAGE with the Proteins on Novex XCell SureLock Mini Cell is provided below IEF Gels 1 Fill the 2D or IPG well with the appropriate 1X SDS Running Buffer 2 Trim the IEF strip to a length of 5 8 5 9 cm for 2D wells or 6 3 6 4 cm for ZOOM IPG wells such that the strip includes the pH regions containing your proteins of interest 3 Transfer the IEF gel strip into the well of a 1 0 mm or 1 5 mm gel cassette as follows e For 1 0 mm Thick Gels Slide the strip into the well using a gel loading tip Avoid trapping air bubbles between the gel strip and the surface of the gel Wet a piece of thick filter paper 5 8 x 4 cm in 1X SDS Running Buffer and use it to push the IEF gel strip down so it makes contact with the surface of the gel see figure below The paper should hold the IEF gel strip in place Filter Paper i i SDS Gel IEF Gel
6. e NuPAGE Novex 4 12 Bis Tris ZOOM Gel e Novex 4 20 Tris Glycine ZOOM Gel The second dimension electrophoresis procedure involves reducing and alkylating the proteins focused on your IPG strip in equilibration buffer loading the strip on your second dimension gel and performing SDS PAGE For 2D separation of Novex IEF Gel strips see page 21 You will need the following items for running ZOOM Gels see pages 63 64 for ordering information on Invitrogen products e 4X NuPAGE LDS Sample Buffer e NuPAGE Sample Reducing Agent e NuPAGE Novex 4 12 Bis Tris ZOOM Gel or Novex 4 20 Tris Glycine ZOOM Gel e Running Buffer depending on your gel type e 0 5 agarose solution e lodoacetamide e Plastic flexible ruler or thin weighing spatula e 15 mL conical tubes e Water bath set at 55 C or 65 C e Protein molecular weight marker Dilute 4X NuPAGE LDS Sample Buffer to 1X with deionized water 2 Add 500 uL of the NuPAGE Sample Reducing Agent 10X to 4 5 mL of the 1X NuPAGE LDS Sample Buffer from Step 1 in a 15 mL conical tube Place one IPG strip in this conical tube for equilibration Incubate for 15 minutes at room temperature Decant the Reducing Solution Prepare 125 mM Alkylating Solution by adding 116 mg of fresh iodoacetamide to 5 mL of 1X NuPAGE LDS Sample Buffer from Step 1 5 Add 5 mL of Alkylating Solution from Step 4 to the conical tube containing the IPG strip Incubate for 15 minut
7. even if they are frozen Reoxidation of samples occur during storage and produce inconsistent results Continued on next page General Guidelines for Preparing Samples and Buffers Continued Running Reduced and Non Reduced Samples Heating Samples High Salt Concentration in Samples Guanidine HCl in Samples Cell Lysates For optimal results we do not recommend running reduced and non reduced samples on the same gel If you do choose to run reduced and non reduced samples on the same gel do not run reduced and non reduced samples in adjacent lanes The reducing agent may have a carry over effect on the non reduced samples if they are in close proximity Heating the sample at 100 C in SDS containing buffer results in proteolysis Kubo 1995 We recommend heating samples for denaturing electrophoresis reduced or non reduced at 85 C for 2 5 minutes for optimal results Do not heat the samples for non denaturing native electrophoresis or Zymogram Gels High salt concentrations result in increased conductivity that affects protein migration and can result in gel artifacts in adjacent lanes containing samples with normal salt concentrations Perform dialysis or precipitate and resuspend samples in lower salt buffer prior to electrophoresis Samples solubilized in guanidine HCl have high ionic strength and produce increased conductivity similar to high salt concentrations In addition guanidine precipitates in the
8. 63 for ordering information on pre mixed buffers See pages 65 71 for recipes if you are preparing your own buffers Gel Type Running Buffer Sample Buffer Novex Tris Glycine Gels SDS PAGE Tris Glycine SDS Running Buffer 10X Tris Glycine SDS Sample Buffer 2X Novex Tris Glycine Tris Glycine Native Running Buffer Tris Glycine Native Sample Buffer Gels Native P AGE 10X 2X Novex Tricine Gels Tricine SDS Running Buffer 10X Tricine SDS Sample Buffer 2X Novex Zymogram Gels Tris Glycine SDS Running Buffer 10X Tris Glycine SDS Sample Buffer 2X IEF Gels IEF Cathode Buffer 10X IEF Sample Buffer 2X IEF Anode Buffer 50X TBE Gels TBE Running Buffer 5X Hi Density TBE Sample Buffer 5X TBE Urea Gels TBE Running Buffer 5X TBE Urea Sample Buffer 2X Prep TBE Urea Sample Buffer 2X for preparative gels DNA Retardation Gels TBE Running Buffer 5X Hi Density TBE Sample Buffer 5X Reducing Agent 10 When preparing samples for reducing gel electrophoresis any of the following reducing agents may be used e NuPAGE Reducing Agent see page 63 for ordering information e Dithiothreitol DTT 50 mM final concentration e PB mercaptoethanol 2 5 final concentration e tris 2 carboxyethyl phosphine TCEP 50 mM final concentration Add the reducing agent to the sample up to an hour before loading the gel Avoid storing reduced samples for long periods
9. Compatibility Coomassie Blue 100 500 ng Tris Glycine Bis Tris General Coomassie Fluor Orange 8 16 ng Tricine native Colloidal Coomassie Blue lt 10 ng TM SimplyBlue SafeStain SilverXpress TM SilverQuest SYPRO Ruby Pro Q Diamond Pro Q Emerald Ethidium Bromide SYBR Green 5 ng 1ng Tris Glycine Bis Tris Low sample quantity Tricine TBE Nucleic acid 0 3 2 5 ng Bis Tris Tricine TBE 0 3 0 9 ng 50 bp 0 25 1 ng Tris Glycine Bis Tris Low sample quantity Tricine native Nucleic acid Mass Spec 60 pg dsDNA Nucleic acid 100 300 pg ssDNA 1 2 ng 24 bp Methods General Guidelines for Preparing Samples and Buffers Introduction Recommended Buffers The XCell SureLock Mini Cell and a power supply are needed to perform electrophoresis with Novex Pre Cast gels Additional reagents supplied by the user are described for each individual protocol General guidelines for preparing samples and buffers for Novex Pre Cast gels are discussed below Detailed instructions for preparing the sample buffer and running buffer are described in the sections for each individual type of gel The recommended running buffer and sample buffer for each Novex Pre Cast Gel is listed in the table below Prepare your sample in the appropriate sample buffer such that the final concentration of the sample buffer is 1X Running buffer must be diluted to 1X final concentration before use See page
10. DNA Retardation Gels Ordering information for pre mixed buffers is on page 63 If you are preparing your own buffers recipes are provided on pages 69 70 e DNA sample e Deionized water e Novex Hi Density TBE Sample Buffer e Novex TBE Running Buffer 1 Prepare samples for DNA Retardation Gels as described below Reagent Amount Sample x pL Novex Hi Density TBE Sample Buffer 5X 1 uL Deionized Water to 9 uL Total Volume 10 pL 2 Load the samples immediately on the gel Specific buffer conditions may be required during incubation of the protein and DNA target sequence in order to minimize non specific DNA protein interactions for certain samples If salt concentration is low 0 1 M or less the samples can usually be loaded in the incubation buffer after adding about 3 5 glycerol and a small amount of bromophenol blue tracking dye Continued on next page DNA Retardation Gels Continued Preparing Running Prepare 1 000 mL of 0 5X Novex TBE Running Buffer as follows Buffer 1 Reagent Amount Novex TBE Running Buffer 5X 100 mL Deionized Water 900 mL Total Volume 1 000 mL 2 Mix thoroughly Use this buffer to fill the Upper and Lower Buffer Chamber of the XCell SureLock Mini Cell for electrophoresis Electrophoresis See page 32 instructions for running DNA Retardation Gels using the XCell Conditions SureLock Mini Cell Run the gel at 100 V constant See page 33 for additional details on electrophoresi
11. Gel Tension Wedge Refer to the XCell SureLock Mini Cell manual IM 9003 for detailed instructions Note If you are running just one gel use the plastic Buffer Dam in place of the second gel cassette to form the Upper Buffer Chamber 6 Fill the Upper Buffer Chamber with a small amount of the Running Buffer to check for tightness of seal If you detect a leak from Upper to the Lower Buffer Chamber discard the buffer reseal the chamber and check the seal again 7 Once the seal is tight fill the Upper Buffer Chamber Inner with the appropriate 1X Running Buffer The buffer level must exceed the level of the wells 8 Load an appropriate volume of sample at the desired protein concentration onto the gel see page 8 for recommended loading volumes 9 Load appropriate protein molecular weight markers see page 64 for ordering information 10 Fill the Lower Buffer Chamber with 600 mL of the appropriate 1X Running Buffer 11 Place the XCell SureLock Mini Cell lid on the Buffer Core With the power on the power supply turned off connect the electrode cords to the power supply red to jack black to jack 12 See next page for Electrophoresis Conditions Continued on next page 32 Power Supply Settings for Novex Pre Cast Gels Electrophoresis Run your gels according to the following protocol Conditions Gel Type Voltage Expected Run Time Current Tris Glycine Gels 125 V constant Start 30 40mA_ 90
12. bands migrate within the shaded regions Tris Glycine Gels Large Proteins Mid Size Proteins Small Proteins Wide Range 116 500 kDa 20 250 kDa 3 60 kDa 6 200 kDa 4 12 8 16 4 20 10 20 10 PEt TEE EE 200 kDa Ze 200ikDa 16 kDa l a kDa 200 kDa 97 kDa So E Owe 116 ETNE Lid 200 kDa e 200 kDa m a kDa AE AAA oi MN 4 6 pa kDa M6 kDa kDa 200 kDa 30 16 kDa 55 kDa Etica kDa kDa kDa 40 E esi 7 2 koa 36 kDa 50 DA 97 reg ER 200 kDa ET ii 31 kDa 60 55 kDa 31 kDa AD eea 36 kDa E E E i E E 31 kDa Y kDa 97 oM 6 kDa 55 36 kDa sa 36 kDa a Y kDa 80 4 Taka 6 kDa 36 kDa 6 oo 6 kDa 90 31 kDa E EEE is co 16 kDa E 5 kDa 6 E of length of gel 100 55 kDa 36 kDa Bands correspond to the migration of Mark12 Unstained Standard under denaturing conditions Continued on next page 72 Gel Migration Charts Continued Novex Tricine IEF The migration patterns of protein markers on Novex Tricine IEF and and Zymogram Gel Zymogram Gels are shown on the table below Optimal resolution is achieved Migration Chart when protein bands migrate within the shaded regions Tricine Gels Peptides IEF Gels pl Zymogram Gels Proteases Synthetic Isoelectric Point i Blotting amp Peptides i Sob AG Protease Analysis Sequencing amp Tryptic nae Analysis 4 16 Gel 10 Gel 12 Gel w pre stained w gelatin w casein c
13. be trapped between the paper gel and plastic wrap by rolling a small glass pipette over the gel Use additional gel drying solution to help remove the air bubbles Use Proper Gel Dryer Set up Place gel on the gel dryer with the plastic wrap facing up Make sure the vacuum pump is in working condition and properly set up to form a tight seal when on Use drying conditions for polyacrylamide gels with the temperature increasing to a set value and holding for the duration of the drying cycle We recommend drying mini gels at 80 C for 2 hours Ensure Gel is Completely Dry The gel will crack if the vacuum seal of the heated gel dryer is broken prior to complete drying of the gel To ensure the gel is completely dried before releasing the vacuum seal follow these tips e Check the temperature of the gel The temperature of the dried gel should be the same as the temperature of the surrounding gel drying surface If the temperature of the dried gel is cooler then the gel is not completely dried e Check for moisture in the tubing connecting the gel dryer to the vacuum pump The gel is not completely dried if there is residual moisture in the tubing and additional drying time is required 51 Blotting Novex Pre Cast Gels Introduction Power Considerations for Blotting Materials Supplied by the User Preparing Transfer Buffer 52 After performing electrophoresis proteins can be transferred to membranes for subsequen
14. bottom of the gel 75 minutes Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel 90 minutes Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel 33 Opening Novex Pre Cast Gel Cassettes Removing the Gel after Electrophoresis 34 After electrophoresis is complete shut off the power disconnect electrodes and remove gel s from the XCell SureLock Mini Cell Separate each of the three bonded sides of the cassette by inserting the Gel Knife into the gap between the two plastic plates that make up the cassette The notched well side of the cassette should face up Push down gently on the knife handle to separate the plates Repeat on each side of the cassette until the plates are completely separated Caution Use caution while inserting the Gel Knife between the two plates to avoid excessive pressure on the gel Carefully remove and discard the top plate allowing the gel to rest on the bottom slotted plate If blotting proceed to page 52 without removing the gel from the bottom plate If staining remove the gel from the plate by one of the methods e Use the sharp edge of the Gel Knife to remove the gel foot from the bottom of the gel Hold the Gel Knife at a 90 angle perpendicular to the gel and the slotted half of the cassette Push down on the knife and then repeat the motion across the gel to cut off the entire foot Hold the plat
15. buffer to fill the Upper and Lower Buffer Chambers of the XCell SureLock Mini Cell for electrophoresis Novex Tricine Gel are not compatible with buffers for Tris Glycine gels e Samples run in Tris Glycine SDS Sample Buffer are poorly resolved e Samples run in Tris Glycine SDS Running Buffer take longer to complete and result in poor resolution of smaller proteins Protein samples for Tricine Gels can be denatured or denatured and reduced 1 Prepare each reduced or non reduced samples for running on Tricine gels as described below Reagent Reduced Non reduced Sample Sample Sample x pL x pL Novex Tricine SDS Sample Buffer 2X 5uL 5uL NuPAGE Reducing Agent 10X 1uL Deionized Water to 4 uL to 5 pL Total Volume 10 pL 10 pL 2 Heat samples at 85 C for 2 minutes Load the samples onto the gel immediately Note For reduced sample add the reducing agent immediately prior to electrophoresis to obtain the best results Leave an empty lane between samples with and without reducing agent to prevent diffusion of the reducing agent into non reduced sample lanes See page 32 for instructions on running Novex Pre Cast Gels using the XCell SureLock Mini Cell Run the gel at 125 V constant See page 33 for additional details on electrophoresis conditions Any of the techniques described on pages 35 46 are suitable for staining Novex Tricine Gels after electrophoresis 15 Zymogram Gels Zymogram Zymogra
16. described on entire sample This effect is page 8 If your sample is too dilute intensified for larger proteins concentrate the sample using salt precipitation or ultrafiltration RNase contamination e Always wear gloves and use sterile techniques to prevent RNase contamination Sample renatured e Heat the sample for 3 minutes at 70 C and keep the sample in ice to prevent renaturation Proceed to electrophoresis immediately after loading Sample overloaded e Recommended DNA load is 0 16 0 33 ug band Urea not completely flushed from e Be sure to thoroughly flush urea the wells out of the wells prior to loading the sample Appendix Accessory Products Electrophoresis Ordering information on a variety of electrophoresis reagents and apparatus Reagents available from Invitrogen is provided below For more information visit our website at www invitrogen com or call Technical Support see page 76 Product XCell SureLock Mini Cell XCell II Blot Module PowerEase 500 Power Supply DryEase Mini Gel Drying System StainEase Staining Tray Gel Dry Drying Solution iBlot Gel Transfer Device Novex Semi Dry Blotter Novex Tris Glycine SDS Running Buffer 10X NuPAGE Sample Reducing Agent 10X NuPAGE LDS Sample Buffer 4X Novex Tris Glycine Transfer Buffer 25X Novex Tris Glycine Native Running Buffer 10X Novex Tris Glycine Native Sample Buffer 2X Novex Tris Glycine SDS Sample Buffer 2X Novex
17. drying We recommend using the DryEase Mini Gel Drying System to air dry the gel A brief gel drying protocol using the DryEase Mini Gel Drying System is provided below For more details on this system refer to the DryEase Mini Gel Drying System manual IM 2380 This manual is available for download from our website at www invitrogen com or contact Technical Support see page 76 1 After all staining and destaining steps are complete wash the destained gel s three times for two minutes each time in deionized water 50 mL per mini gel on a rotary shaker 2 Decant the water and add fresh Gel Dry Drying Solution 35 mL per mini gel 3 Equilibrate the gel in the Gel Dry Drying Solution by shaking the gel for 15 20 minutes in the StainEase Gel Staining Tray or in a round container Note Do not equilibrate gels stained with Coomassie G 250 in the Gel Dry Drying Solution for more than 5 minutes to avoid losing band intensity 4 Cutany rough edges off the gel including the wells and the gel foot using the Gel Knife or a razor blade Remove 2 pieces per gel of cellophane from the package TM Immerse one sheet at a time in the Gel Dry Drying Solution Allow 10 seconds for complete wetting before adding additional sheets Do not soak the cellophane for more than 2 minutes Continued on next page 49 Gel Drying Continued DryEase Mini Gel Drying System continued 50 7 10 11 12
18. fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com SDS Safety Data Sheets SDSs are available at www invitrogen com sds Certificate of The Certificate of Analysis provides detailed quality control and product Analysis qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Continued on next page 76 Technical Support Continued Limited Warranty Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen produ
19. flat for scanning photography display and overhead projection Continued on next page Gel Drying Continued Vacuum Drying General guidelines are provided below to minimize cracking during vacuum drying of gels For detailed instructions on vacuum drying follow the manufacturer s recommendations Handle Gels with Care Remove the gel from the cassette without breaking or tearing the edges Small nicks or tears can act as a starting point for cracking Remove the gel wells and foot off the bottom of the gel with a Gel Knife or a razor blade as described on page 34 Use the StainEase Staining Tray for staining and destaining gels This tray is designed to facilitate the solution changing process without handling of gels Use a Gel Drying Solution We recommend equilibrating the gel in a gel drying solution such as Gel Dry Gel Drying Solution for 10 30 minutes at room temperature with gentle shaking on an orbital shaker before drying the gel Gel Dry Gel Drying Solution contains a proprietary non glycerol component to effectively regulate the rate of drying and prevent cracking The gel drying solutions do not interfere with autoradiography To prepare your own gel drying solution prepare a solution containing 30 methanol and 5 glycerol Note Do not incubate gels stained with Coomassie G 250 in gel drying solution for more than 5 minutes as the bands may fade Remove Air Bubbles Remove any air bubbles that may
20. gels only Ultrapure water 18 megohm cm recommended Rotary shaker Powder free latex or vinyl gloves Microwave oven 700 1200 W optional Water bath set at 80 C optional General considerations for the protocol include the following Perform all fixation staining and washing steps with continuous gentle agitation e g on an orbital shaker at 50 rpm We recommend polypropylene or polycarbonate containers for staining Glass dishes are not recommended Staining containers should be meticulously clean to minimize contamination and other artifacts For convenience gels may be left in fix solution overnight or longer For convenience gels may be left in SYPRO Ruby stain indefinitely without overstaining although speckling artifacts tend to increase over time As with any fluorescent stain cover the gel container during staining and subsequent wash steps to exclude light Continued on next page SYPRO Ruby Staining Continued Preparing Solutions for SYPRO Ruby Staining SYPRO Ruby Basic Protocol Visualization of SYPRO Ruby Stained Gels Prepare the reagents as described below If you are staining two gels double the reagent volumes Increase volumes 1 5 fold for 1 5mm thick gels e Fix Solution Methanol 100 mL Glacial Acetic Acid 14 mL Ultrapure water to 200 mL e Fix Solution for IEF Gels Methanol 40 mL Trichloroacetic Acid 10g Ultrapure water to 100 mL e Wash Solution Methanol 10 m
21. methods the proteins will be sufficiently unfolded to expose hydrophobic sites and bind more efficiently to the membrane 57 Calibrating Protein Molecular Weight Introduction Protein Secondary Structure Buffer Systems 58 The molecular weight of a protein can be determined based upon its relative mobility by constructing a standard curve with protein standards of known molecular weights The protein mobility in SDS PAGE gels is dependent on the e Length of the protein in its fully denatured state e SDS PAGE buffer systems e Secondary structure of the protein An identical molecular weight standard may have slightly different mobility resulting in different apparent molecular weight when run in different SDS PAGE buffer systems If you are using the Novex protein molecular weight standards see the apparent molecular weights of these standards on the Novex Pre Cast Gels listed on the next page to determine an apparent molecular weight of your protein When using SDS PAGE for molecular weight determination slight deviations from the calculated molecular weight of a protein calculated from the known amino acid sequence can occur due to the retention of varying degrees of secondary structure in the protein even in the presence of SDS This phenomenon is observed in highly organized secondary structures such as collagens histones or highly hydrophobic membrane proteins and in peptides where the effect of local seco
22. minutes dependent on gel type SDS PAGE End 8 12 mA Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel Tris Glycine Gels 125 V constant Start 6 12 mA 1 12 hours Native PAGE End 3 6 mA Tricine Gels 125 V constant Start 80 mA 90 minutes dependent on gel type End 40 mA Run the gel until the phenol red tracking dye reaches the bottom of the gel Zymogram Gels 125 V constant Start 30 40 mA 90 minutes dependent on gel type End 8 12 mA Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel IEF Gels 100 V constant 1 hour Start 5 mA 2 5 hours 200 V constant 1 hour End 6 mA TBE Gels 6 TBE Urea Gels 10 TBE Urea Gels 15 TBE Urea Gels DNA Retardation Gels 500 V constant 30 min 200 V constant 180 V constant 180 V constant 180 V constant 100 V constant Start 10 18 mA End 4 6 mA Start 19 mA End 14 mA Start 15 mA End 8 mA Start 13 mA End 6 mA Start 12 15 mA End 6 15 mA Expected start and end current values are stated for single gels Voltages up to 250 V may be used to reduce the run time 30 90 minutes dependent on gel type Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel 50 minutes Run the gel until the bromophenol blue tracking dye reaches the bottom of the gel 60 minutes Run the gel until the bromophenol blue tracking dye reaches the
23. multiunit proteases migrate as a single unit that can be renatured after electrophoresis 1 Prepare each sample as described below Reagent Amount Sample x pL Novex Tris Glycine SDS Sample Buffer 2X 5 pL Deionized Water to 5 uL Total Volume 10 uL 2 Load the samples onto the gel immediately Do not heat samples for Zymogram Gels See page 32 for instructions on running Novex Pre Cast Gels using the XCell SureLock Mini Cell Run the gel at 125 V constant See page 33 for additional details on electrophoresis conditions After completing electrophoresis renature the enzyme and develop the Zymogram Gels to detect protease activity Requirements for the volume of Zymogram Renaturing Buffer and Zymogram Developing Buffer may vary depending upon the size of your developing tray Up to two mini gels can be treated with every 100 mL of 1X Novex Zymogram Renaturing Buffer 1 Prepare 100 mL of Renaturing Buffer as described below Reagent Amount Novex Zymogram Renaturing Buffer 10X 10 mL Deionized Water 90 mL Total Volume 100 mL 2 Mix thoroughly before use Continued on next page 17 Zymogram Gels Continued Preparing Developing Buffer Developing Zymogram Gels Staining Zymogram Gels 18 Up to two mini gels can be treated with every 100 mL of 1X Novex Zymogram Developing Buffer 1 Prepare 100 mL of Developing Buffer as described below Reagent Amount Novex Zymogram Developing
24. protein sequencing If you are performing protein sequencing use the NuPAGE Transfer Buffer or the 0 5X TBE Running Buffer to perform blotting The NuPAGE Transfer Buffer protects against modification of the amino acid side chains and is compatible with N terminal protein sequencing using Edman degradation Use about 700 mL of 1X Transfer Buffer to soak the pads until saturated Remove the air bubbles by squeezing the pads while they are submerged in buffer Removing the air bubbles is essential as they can block the transfer of biomolecules if they are not removed Cut the transfer membrane and filter paper to the dimensions of the gel or use Novex pre cut membrane filter paper sandwiches e PVDF membrane Pre wet PVDF membrane for 30 seconds in methanol ethanol or isopropanol Briefly rinse in deionized water then place in a shallow dish with 50 mL of 1X Transfer Buffer for several minutes e Nitrocellulose Place the membrane directly into a shallow dish containing 50 mL of 1X Transfer Buffer for several minutes e Filter paper Soak the filter paper briefly in 1X Transfer Buffer immediately prior to use e Gel Use the gel immediately following the run Do not soak the gel in transfer buffer Continued on next page 53 Blotting Novex Pre Cast Gels Continued Western Transfer Wear gloves while performing the blotting procedure to prevent contamination Using the XCell II of gels and membranes and exposure to i
25. supplement the Gel Instruction Cards IM 6000 to IM 6008 supplied with the pre cast gels Complete protocols for sample and buffer preparation electrophoresis conditions staining and blotting are provided in this guide To request the instruction cards or for additional information call Technical Support see page 76 or download the manuals from our website at www invitrogen com Store Novex Pre Cast Gels at 4 C The gels have a shelf life of 4 8 weeks depending upon the gel type when stored at 4 C Do not freeze Novex Pre Cast Gels Use gels immediately from the refrigerator Extended exposure of the gels to room temperature significantly impairs the performance of the gel The Novex Pre Cast Gels are supplied as 10 gels per box Gels are individually packaged in clear pouches with 4 10 mL of Packaging Buffer The Packaging Buffer contains 0 02 sodium azide and residual acylamide monomer Wear gloves at all times when handling gels Warning This product contains a chemical acrylamide known to the state of California to cause cancer Refer to the Invitrogen website for the SDS see page 76 For research use only Not intended for human or animal diagnostic or therapeutic uses Overview of Electrophoresis Introduction Support Matrix Polyacrylamide Gel Electrophoresis PAGE Electrophoresis is a simple rapid and sensitive analytical tool for separating proteins and nucleic acids based on their physica
26. the smaller the pore size allowing resolution of low molecular weight molecules and vice versa Continued on next page Overview of Electrophoresis Continued Buffer Systems Electrophoresis Sample Conditions Electrophoresis is performed using continuous or discontinuous buffer systems Continuous buffer systems utilize a single buffer for the gel and the running buffer Discontinuous buffer systems Ornstein 1964 utilize different gel buffers and running buffer In addition two gel layers of different pore size the stacking and separating gel are used Electrophoresis using a discontinuous buffer system allows concentration of the sample to a narrow region prior to separation resulting in sharper bands and higher resolution Depending upon the application electrophoresis can be performed under the following conditions Denaturing Electrophoresis is performed under denaturing conditions using an anionic detergent such as sodium dodecylsulfate SDS SDS denatures and unfolds the proteins by binding the hydrophobic portions of the protein at a ratio of 1 4 g SDS per gram of protein The resultant SDS protein complexes are highly negatively charged and migrate through the gel based on their size rather than charge Non Denaturing Native Electrophoresis is performed under non denaturing native conditions using buffer systems that maintain the native protein conformation cohesion of subunits and biological activity D
27. 5X 200 mL Deionized Water 800 mL Total Volume 1 000 mL Mix thoroughly Use this buffer to fill the Upper and Lower Buffer Chamber of the XCell SureLock Mini Cell for electrophoresis Continued on next page TBE Gels Continued Preparing Samples Novex TBE Gels require only 10 of the amount of sample used on large gels or agarose gels Dilute your standards and samples to 0 01 OD 0 2 ng band Electrophoresis Conditions Migration of the Dye Fronts Staining the Gel to avoid overloading the gel 1 Prepare samples for TBE gels as described below Reagent Sample Novex Hi Density TBE Sample Buffer 5X Deionized Water Amount x pL 2 pL to 8 uL Total Volume 2 Load the samples immediately on the gel 10 pL See page 32 instructions for running TBE Gel using the XCell SureLock Mini Cell Run the gel at 200 V constant See page 33 for additional details on electrophoresis conditions The size of the DNA fragments visualized at the dye fronts of the different TBE Gels is shown in the table below Gel Type Dye Front 6 TBE Gel 65 bp 8 TBE Gel 25 bp 10 TBE Gel 35 bp 20 TBE Gel 15 bp 4 12 TBE Gel 35 bp 4 20 TBE Gel 25 bp accuracy is 5 bp Novex TBE Gels can be stained by silver staining ethidium bromide and 250 bp 220 bp 120 bp 50 bp 400 bp 300 bp Bromophenol Blue dark blue Xylene Cyanol blue green SYBR Green staining techniques after electrophoresi
28. 6 kDa 7 kDa Insulin 4kDa 4kDa 60 Troubleshooting Introduction Review the information below to troubleshoot your experiments with Novex Gels Observation Run taking longer time Low or no current during the run Faint shadow or ghost band below the expected protein band Streaking of proteins Bands in the outer lane of the gel are curving upwards Cause Running buffer too dilute Incomplete circuit Ghost bands are caused due to a slight lifting of the gel from the cassette resulting in trickling of some sample beyond its normal migration point Gel lifting off the cassette is caused due to e Expired gels e Improper storage of gels e Sample overload e High salt concentration in the sample e Sample precipitates e Contaminants such as membranes or DNA complexes in the sample e Concentrated buffer used e Expired gels used e High voltage used Solution Make fresh running buffer as described in this manual and avoid adjusting the pH of the 1X running buffer Remove the tape from the bottom of the cassette prior to electrophoresis Make sure the buffer covers the sample wells Check the wire connections on the buffer core to make sure the connections are intact Avoid using expired gels Use fresh gels Store the gels at the appropriate temperature see page v Load the appropriate amount of protein as described on page 8 Decrease the salt concentr
29. 6 months when stored at room temperature 4 For blotting dilute this buffer as described on page 52 The pH of the 1X solution is 8 3 Do not use acid or base to adjust the pH Continued on next page Recipes Continued Tricine SDS Sample Buffer Tricine SDS Running Buffer 10X Zymogram Renaturing Buffer The Tricine SDS Sample Buffer is available from Invitrogen see page 63 450 mM Tris HCl 12 Glycerol 4 SDS 0 0025 Coomassie Blue G 0 0025 Phenol Red pH 8 45 1 To prepare 10 mL of 2X Tricine SDS Sample Buffer mix the following reagents 3 M Tris HCl pH 8 45 3 mL Glycerol 2 4mL SDS 0 8 g 0 1 Coomassie Blue G 0 5 mL 0 1 Phenol Red 0 5 mL Mix well and adjust the volume to 10 mL with ultrapure water Store at 4 C The buffer is stable for 6 months when stored at 4 C The Tricine SDS Running Buffer is available from Invitrogen see page 63 100 mM Tris base 100 mM Tricine 0 1 SDS pH 8 3 1 To prepare 1 000 mL of 10 x Tricine SDS Running Buffer dissolve the following reagents in 900 mL deionized water Tris Base 121 g Tricine 179 g SDS 10 g Mix well and adjust the volume to 1 000 mL with ultrapure water Store at room temperature The buffer is stable for 6 months when stored at room temperature 4 For electrophoresis dilute this buffer to 1X with water see page 15 The pH of the 1X solution is 8 3 Do not use acid or base to adjust the pH The Zymogram Renaturing Buffer is avail
30. Buffer 10X 10 mL Deionized Water 90 mL Total Volume 100 mL 2 Mix thoroughly before use Note Gels will be treated with Developing Buffer twice so additional buffer may be required depending upon the size of the developing tray 1 Remove the gel from the cassette or remove the top gel plate and allow the gel to remain on the bottom gel plate for support 2 Incubate the gel in 1X Novex Zymogram Renaturing Buffer for 30 minutes at room temperature with gentle agitation 3 Decant the Zymogram Renaturing Buffer and add 1X Novex Zymogram Developing Buffer to the gel 4 Equilibrate the gel for 30 minutes at room temperature with gentle agitation 5 Decant the Developing Buffer and add fresh 1X Novex Zymogram Developing Buffer to the gel 6 Incubate the gel at 37 C for at least 4 hours or overnight for maximum sensitivity The incubation time can be reduced to 1 hour for concentrated samples The optimal result is determined empirically by varying the sample load or incubation time Zymogram Blue Casein 4 16 gels do not require staining For non pre stained Zymogram gels stain the gels with Colloidal Blue Staining Kit or the SimplyBlue Safestain as described on pages 36 37 Areas of protease activity appear as clear bands against a dark background IEF Gels Isoelectric Focusing IEF 2D Electrophoresis Power Considerations for IEF Isoelectric focusing IEF is an electrophoretic technique for t
31. E Urea Gels DNA RNase Oligo Large E Retar Protection Oligos Size dation Assays 9 Check 6 6 10 15 2645 aa 1605 a HE bp 517 pr m 460 bp 350 bp S 58 26 bp So 30 75 z 36 bp m Ss Values are given in bases Continued on next page Gel Migration Charts Continued ZOOM Gel Migration Chart the shaded regions of length of gel The migration patterns of protein standards on ZOOM Gels are shown on the table below Optimal resolution is achieved when protein bands migrate within Zoom Gels 4 12 4 12 4 20 Bis Tris Bis Tris Tris Glycine Zoom Gel Zoom Gel Zoom Gel w MES w MOPS Running Running Buffer Buffer 200 kD 20 200 kDa 30 16 kDa Say 97 kDa Za 55 kDa ES 36 kD 70 31 kDa so 3 k 3 5 kDa Y kD 90 On ZOOM Gels migration of bands correspond to the migration of Mark12 Unstained Standard Cat no LC5677 under denaturing conditions 75 Technical Support World Wide Web Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes SDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write
32. L Glacial Acetic Acid 7 mL Ultrapure water to 100 mL The basic protocol results in the maximum signal strength and widest linear dynamic range for staining of denaturing gels non denaturing gels and IEF gels Sensitivity is in the 1 ng range for most proteins 1 After electrophoresis place the gel into a clean container with 100 mL of Fix Solution and agitate on an orbital shaker for 30 minutes Pour off the used fix solution and repeat once more with fresh Fix Solution Note For IEF Gels place the gel into a clean container with 100 mL of IEF Fix Solution and agitate on an orbital shaker for 3 hours After fixing perform 3 washes in ultrapure water for 10 minutes each before proceeding to the staining step Pour off the used fix solution Add 60 mL of SYPRO Ruby gel stain to the tray containing the gel Agitate on an orbital shaker overnight 4 Transfer the gel to a clean container and wash in 100 mL of Wash Solution for 30 minutes The transfer step helps minimize background staining irregularities and stain speckles on the gel 5 Rinse the gel in ultrapure water for 5 minutes Repeat the rinse a minimum of one more time to prevent possible corrosive damage to your imager Note If you are staining two gels double the reagent volumes Increase volumes 1 5 fold for 1 5mm thick gels Proteins stained with SYPRO Ruby protein gel stain are readily visualized using a UV or blue light source The use of a photographic cam
33. V constant for 1 hour Expected Current Start 39 mA End 35 mA Continued on next page Blotting Novex Pre Cast Gels Continued Blotting IEF Gels Blotting Native Gels Novex IEF Gels are composed of 5 polyacrylamide and are more susceptible to hydrolysis due to the heat generated with the recommended blotting protocol The following protocol has been optimized to prevent hydrolysis and effective transfer of basic proteins due to the low pH of the transfer buffer 1 Prepare chilled 0 7 acetic acid 2 After electrophoresis remove the gel from the cassette and equilibrate the gel in the 0 7 acetic acid for 10 minutes Tip The 5 polyacrylamide gels are stickier and more difficult to handle than higher percentage polyacrylamide gels To prevent the gel from sticking to the filer paper before it is in the proper position remove the gel from the equilibration solution by submerging a piece of filter paper under the gel while it is floating in the equilibration solution When the gel and filter paper are in the correct position lift the filter paper so that it attaches to the gel 3 Assemble the gel membrane sandwich as described on page 54 except in reverse order so that the membrane is on the cathode side of the gel 4 Transfer for 1 hour at 10 V constant During SDS PAGE all proteins have a net negative charge due to the SDS in the sample buffer and the running buffer Proteins separated during native gel elect
34. a 80 kDa Band 5 60 kDa 60 kDa Band 6 50 kDa 50 kDa Band 7 40 kDa 40 kDa Band 8 30 kDa 30 kDa Band 9 20 kDa 20 kDa Band 10 15 kDa 15 kDa Band 11 10 kDa 10 kDa Band 12 3 5 kDa Mark 12 Unstained Standard Tris Glycine Gels 4 20 Tricine Gels 10 20 Myosin 200 kDa 200 kDa P Galactosidase 116 3 kDa 116 3 kDa Phosphorylase B 97 4 kDa 97 4 kDa Bovine Serum Albumin 66 3 kDa 66 3 kDa Glutamic Dehydrogenase 55 4 kDa 55 4 kDa Lactate Dehydrogenase 36 5 kDa 36 5 kDa Carbonic Anhydrase 31 kDa 31 kDa Trypsin Inhibitor 21 5 kDa 21 5 kDa Lysozyme 14 4 kDa 14 4 kDa Aprotinin 6 kDa 6 kDa Insulin B Chain Unresolved Insulin 3 5 kDa Insulin A Chain 2 5 kDa Continued on next page 59 Calibrating Protein Molecular Weight Continued Assigned Apparent Molecular Weights continued SeeBlue Pre Stained Standard Tris Glycine Gel 4 20 Tricine Gel 10 20 Myosin 250 kDa 210 kDa BSA 98 kDa 78 kDa Glutamic Dehydrogenase 64 kDa 55 kDa Alcohol Dehydrogenase 50 kDa 45 kDa Carbonic Anhydrase 36 kDa 34 kDa Myoglobin 30 kDa 23 kDa Lysozyme 16 kDa 16 kDa Aprotinin 6 kDa 7 kDa Insulin 4kDa 4kDa SeeBlue Plus2 Pre Stained Standard Tris Glycine Gel 4 20 Tricine Gel 10 20 Myosin 250 kDa 210 kDa Phosphorylase B 148 kDa 105 kDa BSA 98 kDa 78 kDa Glutamic Dehydrogenase 64 kDa 55 kDa Alcohol Dehydrogenase 50 kDa 45 kDa Carbonic Anhydrase 36 kDa 34 kDa Myoglobin 22 kDa 17 kDa Lysozyme 16 kDa 16 kDa Aprotinin
35. able from Invitrogen see page 63 25 v v Triton X 100 1 To prepare 500 mL of 10X Zymogram Renaturing Buffer add 125 mL Triton X 100 to 300 mL ultra pure water Mix well and adjust the volume to 500 mL with ultrapure water Store at room temperature The buffer is stable for 6 months when stored at room temperature Continued on next page 67 Recipes Continued Zymogram Developing Buffer IEF Sample Buffer pH 3 7 IEF Sample Buffer pH 3 10 68 The Zymogram Developing Buffer is available from Invitrogen see page 63 50 mM Tris base 40 mM HCl 200 mM NaCl 5 mM CaCl 0 02 w v Brij 35 1 To prepare 500 mL of 10X Zymogram Developing Buffer dissolve the following reagents in 400 mL deionized water Tris Base 30 2 g 6N HCl 33 mL NaCl 58 5 g CaCl 2H 0 3 7 8 Brij 35 1 0g Mix well and adjust the volume to 500 mL with ultrapure water Store at room temperature The buffer is stable for 6 months when stored at room temperature 4 For developing the zymogram gel dilute this buffer to 1X with water see page 18 The IEF Sample Buffer pH 3 7 is available from Invitrogen see page 63 40 mM Lysine free base 15 Glycerol 1 To prepare 10 mL of 2X IEF Sample Buffer pH 3 7 mix the following reagents 10X IEF Cathode Buffer pH 3 7 see next page 2mL Glycerol 3 mL 2 Mix well and adjust the volume to 10 mL with ultrapure water Store at 4 C The buffer is stable for 6 months when store
36. ae ea l Blotting Pad a N Blotting Pad l Filter Paper E Transfer Membrane Fa First Gel Cathode Core Filter Paper ETS AA TT Blotting Pad e o o o o o o o o 0 Blotting Pad l Cathode Core Continued on next page 55 Blotting Novex Pre Cast Gels Continued Recommended The transfer conditions for Novex Pre Cast Gels using the XCell IT Blot Transfer Module are listed in the table below Conditions Note The expected current listed in the table is for transferring one gel If you are transferring two gels in the blot module the expected current is roughly twice the listed value Gel Transfer Buffer Membrane Power Conditions 56 Tris Glycine Gel Tricine Gel IEF Gel TBE Gel TBE Urea Gel DNA Retardation Gel 1X Tris Glycine Transfer Buffer with 20 methanol 1X Tris Glycine Transfer Buffer with 20 methanol 0 7 Acetic acid pH 3 0 See next page for details on this alternate transfer protocol 0 5X TBE Running Buffer 0 5X TBE Running Buffer 0 5X TBE Running Buffer Nitrocellulose or PVDF Nitrocellulose or PVDF Nitrocellulose or PVDF 25 V constant for 1 2 hours Expected Current Start 100 mA 25 V constant for 1 hour Expected Current Start 65 85 mA 10 V constant for 1 hour Expected Current Start 65 85 mA 30 V constant for 1 hour Expected Current Start 39 mA End 35 mA 30 V constant for 1 hour Expected Current Start 39 mA End 35 mA 30
37. ale your sample load for Ce O mm 400 nL 12 ug band 2 0 ug band the sensitivity of 15mm 600 HL your silver 10mm 7cmIPGStrip N A N A staining kit For use with the GPP 10mm 60 nL 2 ug 400 ng band SilverQuest or 10mm 28uL 0 5 ug band 100 ng band SilverXpress Silver Staining CS 10mm 25pL 0 5 pg band 100 ng band Kits we 15mm 37 pL recommend a Geel 20 uL 0 5 band 100 band protein load of 10mm 201 1g ban ng ban o 10mm PL 0 5 pg band 100 ng band 15mm 2 BL Choosing the Appropriate Well for Your Application Choose the type of well for your application based upon the volume of your sample The more wells a comb has and the thinner the gel is the lower the sample loading volume Note Proteins transfer out of a 1 0 mm gel more easily than from a 1 5 mm gel Gel Staining Staining Novex Pre Cast Gels The Novex Pre Cast Gels are compatible with most silver staining protocols We recommend using the SilverQuest Silver Staining Kit or the SilverXpress Silver Staining Kit see pages 35 43 for silver staining of Novex Gels Novex Pre Cast Gels are compatible with any of the standard Coomassie staining procedures Protocols that are accelerated by heat are preferable as heat can fix proteins especially smaller peptides The SimplyBlue SafeStain see page 36 and Novex Colloidal Blue Staining Kit see page 37 are recommended for staining Novex Gels Stain Type Sensitivity Gel Type Application
38. as a leading ion due to its high affinity to the anode relative to other anions in the system The gel buffer ions are Tris and Cl pH 8 65 e Glycine is the primary anion in the running buffer and serves as a trailing ion Glycine is partially negatively charged and trails behind the highly charged chloride ions in the charged environment The running buffer ions are Tris Gly and dodecylsulfate pH 8 3 e Tris Base is the common ion present in the gel buffer and running buffer During electrophoresis the gel and buffer ions in the Tris Glycine system form an operating pH of 9 5 in the separation region of the gel The following reagents are needed to perform electrophoresis with Novex Tris Glycine Gels Ordering information for pre mixed buffers is on page 63 If you are preparing your own buffers recipes are provided on pages 65 66 e Protein sample e Deionized water e Protein molecular weight markers For denaturing electrophoresis e Novex Tris Glycine SDS Sample Buffer e NuPAGE Reducing Agent e Novex Tris Glycine SDS Running Buffer For non denaturing native electrophoresis e Novex Tris Glycine Native Sample Buffer e Novex Tris Glycine Native Running Buffer Use 1X Tris Glycine SDS Running Buffer for electrophoresis of denatured samples or 1X Native Running Buffer for electrophoresis of native samples 1 Prepare 1 000 mL of Running Buffer as described below Reagent Amount 10X Nove
39. asein blue 20 36 kDa 16 kDa 30 __ 97 kDa 31 kDa 40 50 of length of gel 60 70 80 100 Bands correspond to the migration of Mark12 Unstained Standard under denaturing conditions The numbered bands on the Zymogram Gel patterns refer to the following proteases Band 1 Collagenase Type 140 kDa Band 2 Thermolysin 37 kDa Band 3 Chymotrypsin 30 kDa Band 4 Trypsin 19 kDa Continued on next page 73 Gel Migration Charts Continued Novex TBE and TBE Urea Gel Migration Chart 74 The migration patterns of DNA fragments on Novex TBE and TBE Urea Gels are shown on the table below Optimal resolution is achieved when nucleic acid bands migrate within the shaded regions TBE Gels Restriction Digest PCR Products Restriction Digest Oligo PCR Products nucleotides 4 12 6 8 10 20 4 20 10 ae 2645 bp 2645 bp 2645 rara E DS 1605 _ 2645 1198 pes 1605 b 20 z ooe MN 1605 ES Geil RBA 198bp 198 bp 1605 or 1605 oa 198 Toe bp 30 Ci 7b ERA 40 50 ET 460bp 350 bp EA en 350 bp a 517 E E 50 179 bp 460 we M517 bp o oo Er pi ears A D Ea 460bp 350bp 5 E A E 60 n E ESO ETE 179 ET 26 bp 51 bp te MS 26 Erm 20 36 PSSS y 75 75 bp 179 n 51 30 21 Paes 26 ETE 36 ES 51 mi 75 bp 36 75 bp 90 36 51 bp 51 bp Values are given in base pairs bp 21 bp TB
40. ases and current decreases over the course of the run Constant power is typically used when using IEF strips When using constant power set the voltage limit slightly above the maximum expected for the run High local resistance can cause a large amount of heat to be generated over a small distance damaging the electrophoresis cell and gels Novex Pre Cast Gels Novex Gel Specifications Introduction Specifications The Novex Pre Cast Gel cassette is 10 cm x 10 cm in size and designed for use with the XCell SureLock Mini Cell and XCell6 MultiGel Unit see page 63 for ordering information Novex Pre Cast Gels are available for resolving proteins in the range of 2 500 kDa and nucleic acids in the range of 10 3 000 bp depending upon the type and acrylamide percentage of the gel Refer to Gel Selection page 6 for details on applications and migration patterns Gel Matrix Gel Thickness Gel Size Cassette Size Cassette Material Sample Well Configuration Acrylamide Bisacrylamide 1 0 mm or 1 5 mm 8 cm x 8 cm 10 cm x 10 cm Styrene Copolymer recycle code 7 1 5 9 10 12 15 well 2D well and IPG well Continued on next page Novex Gel Specifications Continued Novex Gel Formulations All Novex Pre Cast gels are made with high purity reagents The gels for DNA analysis are DNase free The composition of the different gels is listed below Gel Type Tris Glycine Gels except 4
41. ation of your sample using dialysis or gel filtration Increase the concentration of SDS in your sample if necessary to maintain the solubility of the protein Centrifuge or clarify your sample to remove particulate contaminants The pre made buffers are supplied as concentrate Dilute the buffers as described in this manual Avoid using gels after the expiration date Electrophorese the gel using conditions described on page 33 Continued on next page 61 Troubleshooting Continued Observation Bands in the outside lanes of the gel smiling Bands are running as U shape rather than a flat band Bands appear to be funneling or getting narrower as they progress down the gel Dumbbell shaped bands after electrophoresis For TBE Urea gels High background and smeared bands or abnormal band shapes 62 Cause Solution Expired gels used causing the Avoid using gels after the acrylamide to break down in the gel expiration date Use fresh gels Samples are loaded on the gel and Load samples on to the gel not electrophoresed immediately immediately before electrophoresis resulting in sample diffusion Proteins are over reduced causing Reduce the proteins using DTT or the proteins to be negatively P mercaptoethanol as described on charged and repel each other page 10 Loading a large volume of sample Load the appropriate volume of causing incomplete stacking of the sample per well as
42. cm over the edge of cathode core Place the anode core on top of the pads The gel membrane assembly should be held securely between the two halves of the blot module ensuring complete contact of all components Position the gel membrane sandwich and blotting pads in the cathode core of the XCell IT Blot Module to fit horizontally across the bottom of the unit There should be a gap of approximately 1 cm at the top of the electrodes when the pads and assembly are in place Hold the blot module together firmly and slide it into the guide rails on the Lower Buffer Chamber The blot module fits into the unit only one way with the sign at the upper left hand corner of the blot module and the inverted gold post fitting into the connector on the right side of the Lower Buffer Chamber Place the gel tension wedge so that its vertical face is against the blot module Lock the gel tension wedge by pulling the lever forward Continued on next page Blotting Novex Pre Cast Gels Continued Western Transfer 13 Using the XCell 11 Fill the blot module with 1X Transfer Buffer until the gel membrane sandwich is covered in Transfer Buffer To avoid generating extra Blot Module conductivity and heat do not fill the chamber all the way to the top continued 14 Fill
43. cts are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 77 References Kubo K 1995 Effect of Incubation of Solutions of Proteins Containing Dodecyl Sulfate on the Cleavage of Peptide Bonds by B
44. d at 4 C The IEF Sample Buffer pH 3 10 is available from Invitrogen see page 63 20 mM Lysine free base 20 mM Arginine free base 15 Glycerol 1 To prepare 10 mL of 2X IEF Sample Buffer pH 3 10 mix the following reagents 10X IEF Cathode Buffer pH 3 10 see next page 2mL Glycerol 3 mL Mix well and adjust the volume to 10 mL with ultrapure water Store at 4 C The buffer is stable for 6 months when stored at 4 C Continued on next page Recipes Continued IEF Cathode Buffer pH 3 7 IEF Cathode Buffer pH 3 10 IEF Anode Buffer TBE Running Buffer The IEF Cathode Buffer pH 3 7 is available from Invitrogen see page 63 40 mM Lysine free base 1 To prepare 100 mL of 10X IEF Cathode Buffer pH 3 7 dissolve 5 8 g of Lysine free base in 100 mL of ultrapure water 2 Store at 4 C The buffer is stable for 6 months when stored at 4 C The IEF Cathode Buffer pH 3 10 is available from Invitrogen see page 63 20 mM Lysine free base 20 mM Arginine free base You can use D L or D L form of arginine pH 10 1 1 To prepare 100 mL of 10X IEF Cathode Buffer pH 3 10 dissolve 2 9 g of Lysine free base and 3 5 g of Arginine free base in 100 mL of ultrapure water 2 Store at 4 C The buffer is stable for 6 months when stored at 4 C The IEF Anode Buffer is available from Invitrogen see page 63 7 mM Phosphoric acid 1 To prepare 100 mL of 50X IEF Anode Buffer mix 2 4 mL of 85 phos
45. e and gel over a container with the gel facing downward and use the knife to carefully loosen one lower corner of the gel and allow the gel to peel away from the plate e Hold the plate and gel over a container with the gel facing downward Gently push the Gel Knife through the slot in the cassette until the gel peels away from the plate Cut the gel foot off of the gel after fixing and staining but before drying Fix and stain the gel as described on pages 35 48 For developing the Zymogram gel for enzyme activity see page 17 For fixing IEF gels see page 21 Coomassie Staining Introduction Note Molecular Weight Calibration Materials Supplied by the User Instructions are provided below for Coomassie staining Tris Glycine Zymogram IEF and Tricine Gels using the SimplyBlue SafeStain Colloidal Blue Staining Kit and Coomassie R 250 If you are using other types of Coomassie staining kits follow the appropriate manufacturer s recommendations If you are staining low molecular weight peptides lt 2 5 kDa we recommend fixing the gel in 5 glutaraldehyde and 50 methanol for one hour and then follow the instructions in the Colloidal Blue Staining Kit Manual IM 6025 for small peptides Guidelines and apparent molecular weight values for Novex protein molecular weight standards are provided on page 59 You will need the following items for staining your gel see page 63 for ordering information on Invitrogen p
46. e fixing step is not required for Tris Glycine Tricine and Zymogram Gels 2 Rinse the mini gel 3 times for 5 minutes with 100 mL deionized water to remove SDS and buffer salts which interfere with binding of the dye to the protein Discard each rinse 3 Stain the mini gel with enough SimplyBlue SafeStain 20 100 mL to cover the gel Stain for 1 hour at room temperature with gentle shaking Bands will begin to develop within minutes After incubation discard the stain Stain cannot be re used Note Gel can be stained for up to 3 hours but after 3 hours sensitivity will decrease If you need to leave the gel overnight in the stain add 2 mL of 20 NaCl w v in water for every 20 mL of stain This procedure will not affect sensitivity 4 Wash the mini gel with 100 mL of water for 1 3 hours The gel can be left in the water for several days without loss of sensitivity There is a small amount of dye in the water that is in equilibrium with the dye bound to the protein so proteins will remain blue 5 To obtain the clearest background for photography perform a second 1 hour wash with 100 mL water Note Sensitivity decreases at this point if the gel is allowed to stay in the water more than 1 day Reduction of free dye in the water favors dissociation of the dye from the protein If you need to store the gel in water for a few days add 20 mL of 20 NaCl 6 For gel drying see page 49 Continued on next page Coomassie Stai
47. e gel at 125 V constant See page 33 for additional details on electrophoresis conditions Any of the techniques described on pages 35 46 are suitable for staining Novex Tris Glycine Gels after electrophoresis 13 Tricine Gels Tricine Buffer System Advantages of Tricine Gels Materials Supplied by the User 14 The Tricine system is a modification of the Tris Glycine discontinuous buffer system see page 12 developed by Schaegger and von Jagow Schaegger and von Jagow 1987 specifically designed for resolving peptides and low molecular weight proteins In the Tris Glycine system proteins are stacked in the stacking gel between the highly mobile leading chloride ion in the gel buffer and the slower trailing glycine ion in the running buffer These stacked protein bands undergo sieving once they reach the separating gel However the resolution of smaller proteins lt 10 kDa is hindered by the continuous accumulation of free dodecylsulfate DS ions from the SDS sample and running buffers in the stacking gel Smaller proteins mix with DS ions in the zone of stacked DS micelles resulting in fuzzy bands and decreased resolution The mixing also interferes with the fixing and staining of smaller proteins To avoid this problem the Tricine system uses a low pH gel buffer and replaces the trailing glycine ion with a fast moving tricine ion in the running buffer The smaller proteins that previously migrated with the stac
48. era or CCD camera and the appropriate filters is essential to obtain the greatest sensitivity Continued on next page 45 SYPRO Ruby Staining Continued Using SYPRO SYPRO Ruby stain can be used to post stain gels stained with other gel stains Ruby Stain as a such as Pro Q Diamond phosphoprotein gel stain Pro Q Emerald 300 Post Stain glycoprotein gel stain Pro Q Sapphire or InVision oligohistidine tag gel stains or Pro Q Amber transmembrane protein gel stain Always use SYPRO Ruby stain last as the SYPRO Ruby signal can dominate the signal from other stains SYPRO Ruby stain does not work well as a post stain for colorimetric stains such as Coomassie and silver stains 46 SYBR Green Staining Introduction Procedure Visualization of SYBR Green Stained Gels The SYBR Green I nucleic acid gel stain is a sensitive stain that can be used to detect DNA in Novex TBE and TBE Urea Gels As little as 20 60 pg of double stranded DNA 100 300 pg of single stranded DNA or 1 2 ng of a synthetic 24 mer can be detected depending upon the wavelength of transillumination General considerations for the protocol include the following e Werecommend polypropylene containers for staining Glass dishes are not recommended Staining containers should be meticulously clean to minimize contamination and other artifacts e SYBR Green I reagent has optimal sensitivity at pH 7 5 8 0 e For convenience ge
49. es at room temperature 6 Decant the Alkylating Solution and proceed immediately to SDS PAGE page 25 Continued on next page ZOOM Gels Continued SDS PAGE Electrophoresis Conditions Staining the Gel A protocol for separating proteins in an IPG strip by SDS PAGE with ZOOM Gels and the XCell SureLock Mini Cell is provided below 1 Prepare 0 5 agarose solution in the appropriate running buffer and keep it warm 55 65 C until you are ready to use the agarose solution 2 Cut the plastic ends of the IPG strip flush with the gel Do not cut off any portions of the gel 3 Slide the IPG strip into the ZOOM Gel well If the molecular weight marker well is bent straighten the well using a gel loading tip 5 Align the IPG strip properly in the ZOOM Gel well using a thin plastic ruler or a weighing spatula Avoid trapping air bubbles between the strip and the gel while sliding the strip into the well 6 Pour 400 pL of 0 5 agarose solution into the ZOOM Gel well to seal the IPG strip in place Make sure the agarose solution does not overflow into the molecular weight marker well See page 32 for instructions on running Novex Pre Cast Gels using the XCell SureLock Mini Cell Note Do not use the ZOOM IPGRunner Core for electrophoresis of the second dimension gel You must use the Buffer Core supplied with the XCell SureLock Mini Cell Perform electrophoresis at 200 V for 40 minutes for NuPAGE No
50. formamide on TBE Urea polyacylamide gels as it will result in fuzzy bands Use 1X Novex TBE Running Buffer to perform electrophoresis 1 Prepare 1 000 mL of Running Buffer as follows 2 Reagent Amount Novex TBE Rumning Buffer 5X 200 mL Deionized Water 800 mL Total Volume 1 000 mL 3 Mix thoroughly Use this buffer to fill the Upper and Lower Buffer Chamber of the XCell SureLock Mini Cell for electrophoresis 4 Flush wells of the gel several times with 1X TBE Running Buffer to remove urea from the wells prior to loading samples to obtain sharp bands Continued on next page TBE Urea Gels Continued Preparing Samples Novex TBE Urea Gels require only 10 of the amount of sample used on Electrophoresis Conditions Migration of the Dye Fronts Staining the Gel large gels or agarose gels Dilute your standards and samples to 0 01 OD 0 2 pg band to avoid overloading the gel 1 Prepare samples for TBE Urea Gels as described below Reagent Amount Sample x pL Novex TBE Urea Sample Buffer 2X 5 pL Deionized Water to 5 uL Total Volume 10 uL Heat samples at 70 C for 3 minutes to denature the samples Load the samples immediately on the gel If the samples are not used immediately place them on ice to prevent renaturation TM See page 32 instructions for running TBE Urea Gel using the XCell SureLock Mini Cell Run the gel at 180 V constant See page 33 for additional details on electrophoresi
51. he separation of proteins based on their pl The pl is the pH at which a protein has no net charge and thus does not migrate further in an electric field IEF Gels are used to determine the isoelectric point pl of a protein and to detect minor changes in the protein due to post translational modifications such as phosphorylation and glycosylation In IEF proteins are applied to polyacrylamide gels IEF Gels or immobilized pH gradient IPG strips containing a fixed pH gradient As the protein sample containing a mixture of proteins migrates through the pH gradient individual proteins are immobilized in the pH gradient as they approach their pl Novex IEF Gels contain 5 polyacrylamide and are used for native applications The pH 3 10 gels have a pI performance range of 3 5 8 5 and the pH 3 7 gels have a pI performance range of 3 0 7 0 Proteins separated on IEF Gels are suitable for use in two dimensional 2D electrophoresis using Novex Tris Glycine or NuPAGE Gels with a 2D well or ZOOM format to separate focused proteins by mass Two dimensional 2D gel electrophoresis is a powerful and sensitive technique for separating and analyzing protein mixtures from biological samples 2D gel electrophoresis is performed in two consecutive steps 1 First dimension separation of proteins using isoelectric focusing Proteins are separated based on their isoelectric point using IEF gels or IPG strips 2 Second dimension separation of p
52. ical Support see page 76 Product Application Quantity Catalog no SimplyBlue Safe Stain A oe 1L LC6060 staining of proteins in polyacrylamide gels m Qs ac Sensitive silver staining of proteins i AS compatible with mass spectrometry 1 Kit LC6070 analysis Colloidal Blue Staining Kit Sensitive colloidal Coomassie G 250 1 Kit LC6025 staining of proteins in polyacrylamide gels Se o iN SilverXpress Silver High sensitivity low background protein 1 Kit LC6100 Staining Kit and nucleic acid silver staining Mark 12 Unstained For estimating the apparent molecular ene LC5677 Standard weight of proteins MagicMark Western For protein molecular weight estimation on 250 pL LC5600 Standard western blots SeeBlue Pre Stained For monitoring the progress of your run Standard and evaluating transfer efficiency aa pe e ink fe ania SeeBlue Plus2 Pre Stained For visualizing protein molecular weight 500 uL LC5925 Standard range and evaluating transfer efficiency Novex Sharp Pre stained For visualizing protein molecular weight Protein Standard range and evaluating transfer efficiency ees IED BenchMark Protein For estimating the apparent molecular 2 x 250 uL 10747 012 Ladder weight of proteins IEF Marker 3 10 For determining the pl of proteins 500 pL 39212 01 Nucleic Acid Markers A large variety of nucleic acid markers are available from Invitrogen Ready Load format pre mixed with loading buffer nucleic ac
53. id markers are also available for your convenience For more information visit our website at www invitrogen com or contact Technical Support see page 76 64 Recipes Tris Glycine SDS Running Buffer Tris Glycine Native Running Buffer The Tris Glycine SDS Running Buffer is available from Invitrogen see page 63 25 mM Tris Base 192 mM Glycine 0 1 SDS pH 8 3 1 To prepare 1 000 mL of 10X Tris Glycine SDS Running Buffer dissolve the following reagents to 900 mL ultrapure water Tris Base 29 g Glycine 144 g SDS 10g Mix well and adjust the volume to 1 000 mL with ultrapure water Store at room temperature The buffer is stable for 6 months when stored at room temperature For electrophoresis dilute this buffer to 1X with water see page 12 The pH of the 1X solution is 8 3 Do not use acid or base to adjust the pH The Tris Glycine Native Running Buffer is available from Invitrogen see page 63 25 mM Tris base 192 mM Glycine pH 8 3 L To prepare 1 000 mL of 10X Tris Glycine Native Running Buffer dissolve the following reagents to 900 mL ultrapure water Tris Base 29 g Glycine 144 g Mix well and adjust the volume to 1 000 mL with ultrapure water Store at room temperature The buffer is stable for 6 months when stored at room temperature For native electrophoresis dilute this buffer to 1X with water see page 12 The pH of the 1X solution is 8 3 Do not use acid or base to adjust the pH C
54. ing see page 49 Silver Staining Introduction Instructions are provided below for silver staining Novex Gels using the SilverQuest Silver Staining Kit and the SilverXpress Silver Staining Kit see page 63 for ordering information If you are using any other silver staining kit follow the manufacturer s recommendations Molecular Weight Guidelines and apparent molecular weight values for Novex protein Calibration molecular weight standards are provided on page 64 Materials Supplied You will need following items for silver staining your gel see page 63 for by the User ordering information on Invitrogen products e Staining container e Rotary Shaker e Ultrapure water gt 18 megohm cm resistance recommended e Teflon coated stir bars e Disposable 10 mL pipettes e Clean glass bottles for reagent preparation e Graduated glass cylinders e Protein molecular weight markers Mark 12 Unstained Standard recommended For SilverQuest Staining e SilverQuest Silver Staining Kit e 30 ethanol made with ultrapure water e 100 ethanol e Fixative 40 ethanol 10 acetic acid made with ultrapure water For SilverXpress Staining e SilverXpress Silver Staining Kit e Methanol e Acetic acid e Sulfosalicylic acid e Trichloroacetic acid TCA Continued on next page 39 Silver Staining Continued Preparing Solutions for SilverQuest Silver Staining 40 For optimal silver staining re
55. ing Kit Manual IM 6070 This manual is available on our website at www invitrogen com or contact Technical Support see page 76 TM Use 100 mL of each solution for each 1 0 mm thick 8 x 8 cm Novex Gel Note You may have to optimize the staining protocol if the dimensions of your gel are not the same as mentioned above Caution Use caution while performing the Fast Staining Protocol using a microwave oven Do not overheat the staining solutions 1 After electrophoresis place the gel in a clean microwaveable staining tray of the appropriate size Rinse the gel briefly with ultrapure water 2 Place the gel in 100 mL of fixative and microwave at high power 700 watts for 30 seconds Remove the gel from the microwave and gently agitate it for 5 minutes at room temperature Decant the fixative 3 Wash the gel with 100 mL of 30 ethanol in a microwave at high power for 30 seconds Remove the gel from the microwave and gently agitate it for 5 minutes at room temperature on a rotary shaker Decant the ethanol 4 Add 100 mL of Sensitizing solution to the washed gel Microwave at high power for 30 seconds Remove the gel from the microwave and place it ona rotary shaker for 2 minutes at room temperature Decant the Sensitizing solution 5 Add 100 mL ultrapure water to the gel Microwave at high power for 30 seconds Remove the gel from the microwave and gently agitate it for 2 minutes at room temperature Decant the water and repeat
56. ing advantages over other methods of storing IEF gels e Indefinite storage without loss of resolution e Easy to manipulate as bands are visible e Confirms quality of first dimension IEF before proceeding to SDS PAGE Continued on next page 21 IEF Gels Continued Materials Supplied by the User Equilibrating the Gel 22 In addition to the appropriate gel with a 2D well or IPG well the following reagents are needed to perform 2D gel electrophoresis with Novex Gels e 20 Ethanol e Sample Buffer depending on your gel type e Running Buffer depending on your gel type e Filter Paper NuPAGE Sample Reducing Agent optional e lodoacetamide optional The SDS in the sample buffer and running buffer for SDS PAGE strips the stain from proteins and resolubilizes the proteins for migration during 2D electrophoresis 1 Incubate the IEF gel in 100 mL 20 ethanol for 10 minutes 2 Cut out the desired lane strip from the IEF gel for SDS PAGE 3 Incubate the strip in 2 mL 2X SDS sample buffer and 0 5 mL ethanol for 3 5 minutes Aspirate the sample buffer and rinse with 1X Running Buffer 4 Proceed Optional Procedure for Reduced Samples 1 Incubate the IEF gel in 100 mL 20 ethanol for 10 minutes 2 Cut out the desired lane strip from the IEF gel for SDS PAGE 3 Incubate the strip in 2 mL 2X SDS sample buffer and 0 5 mL ethanol for 3 5 minutes Aspirate the sample buffer and rinse with 1X Running Buffer
57. invitrogen by technologies Novex Pre Cast Gel Electrophoresis Guide General information and protocols for using Novex pre cast gels Rev date 07 October 2010 Manual part no IM 1002 MANO0003187 Contents General Infotmationssisicsissesssssesesict ssiessesivssestacatesees sotesssisntaadatansestssssinnscssetussesestua teststst ts sabsctans ieinsest oa sesactanaes v Overview of Electrophoresis sssini ei Shs L EEO EA E E nds 1 Novex Pre Cast Gels tison ai a a AE 4 Novex Gel Specifications tit tenet ara ad ed soeate tieN RS ceased 4 Gel Sele CHO ii dl ads 6 Well Vollmer datada 8 CR NOS 9 MethodsS ates cess eee uence exe wnee cena we eaten we nec enue cesisee ue menace au tasd ecedend pea seascesebessatensanecths 10 General Guidelines for Preparing Samples and Buffers ccccccccseesssseneneseseseeeeteeeeeteseseeeeeeeenenenes 10 Tris Glycine Gels wists ash at hate aati tage tiie at a A E EEEa 12 Tricine Gels ites Li EL Aa Ta E adets Lal dees sues liens Ai ai 14 Ly mogra Gelsi senean iets len sab 16 AA Matec h en ili tan dete late ia hie Rea Aen Reese eS ee cares lees os ton abbas beste 19 ZOOM RGIS sissies seh u aga ni n a css 24 IBER zs corr ssicc sth A E A AA 26 TBE Urea Gels iii ito 28 DNA Retardation Gelsin ee r ea n aa eaaa aea E Oena na EA E EA E Beate 30 Electrophoresis of Novex Pre Cast Gels iii E 32 Power Supply Settings for Novex Pre Cast Gels ssssssssesssssssssssnsessnssnsessassusessas
58. izer 5mL Ultrapure water to 200 mL Staining solution Stainer A 5mL Stainer B 5mL Ultrapure water 90 mL Developing Solution Developer 5mL Ultrapure water 95 mL Continued on next page Silver Staining Continued SilverXpress Silver Staining The following staining procedure is for 1 mm thick Novex Gels If you are using 1 5 mm thick Novex Gels double the incubation time Protocol For gel drying see page 49 Note Gels may be stored in the second Sensitizing Solution overnight if desired Step Solution Vol Gel Gel Type Tris Glycine Tricine TBE TBE Urea IEF 1A Fix the gel in Fixing Solution 200mL 10minutes 10 minutes 10 minutes 10 minutes 1B N A N A N A N A 10 minutes 2A Decant the Fixing Solution 100mL 10minutes 30 minutes 10 minutes 30 minutes 2B and incubate the gel two 100 mL 10 minutes 30 minutes 10 minutes 30 minutes changes of Sensitizing Solution 3A Decant the Sensitizing 200mL 5minutes 5 minutes 5 minutes 5 minutes 3B Solution and rinse the gel 200 mL 5 minutes 5 minutes 5 minutes 5 minutes twice with ultrapure water 4 Incubate the gel in Staining 100mL 15minutes 15 minutes 30 minutes 15 minutes Solution 5A Decant the Staining Solution 200mL 5 minutes 5 minutes 5 minutes 5 minutes aa ane tinse Me pee with 200mL 5 minutes 5 minutes 5 minutes 5 minutes ultrapure water 6 Incubate the gel in 100mL 3 15 minutes 3 15 3 15 minutes 3 15 Developing Solution minutes minutes 7 Add the Stopping Soluti
59. ked DS micelles in the Tris Glycine system become well separated from DS ions in the Tricine system resulting in more efficient stacking and destacking of low molecular weight proteins sharper bands and higher resolution The Tricine Gels have the following advantages over the Tris Glycine Gels for resolving proteins in the molecular weight range of 2 20 kDa e Allows resolution of proteins with molecular weights as low as 2 kDa e Ideal for direct sequencing of proteins after transferring to PVDF as tricine does not interfere with sequencing e Minimizes protein modification because of a lower pH The following reagents are needed to perform electrophoresis with Novex Tricine Gels Ordering information for pre mixed buffers is on page 63 If you are preparing your own buffers recipes are provided on page 67 e Protein sample e Deionized water e Protein molecular weight markers e Novex Tricine SDS Sample Buffer e NuPAGE Reducing Agent for reduced samples e Novex Tricine SDS Running Buffer Continued on next page Tricine Gels Continued Preparing Running Buffer Note Preparing Samples Electrophoresis Conditions Staining the Gel Use 1X Novex Tricine SDS Running Buffer for electrophoresis of Tricine gels 1 Prepare 1 000 mL of Running Buffer as described below Reagent Amount Novex Tricine SDS Running Buffer 10X 100 mL Deionized Water 900 mL Total Volume 1 000 mL 2 Mix thoroughly Use this
60. l characteristics mass isoelectric point etc Most biological molecules carry a net charge at any pH other than their isoelectric point and migrate at a rate proportional to their charge density in an electrical field The mobility of a biological molecule through an electric field depends on the following factors e Field strength e Net charge on the molecule e Size and shape of the molecule e Tonic strength e Properties of the medium through which the molecules migrate e g viscosity pore size Polyacrylamide and agarose are two types of support matrices used in electrophoresis The support matrix is a porous media that acts as a molecular sieve The sieving function depends on the pore size and concentration of the matrix Agarose has a large pore size and is ideal for separating macro molecules such as nucleic acids and protein complexes Polyacrylamide has a smaller pore size and is ideal for separating proteins and smaller nucleic acids Polyacrylamide gels are formed by the polymerization of acrylamide monomers into long chains crosslinked by bifunctional compounds such as N N methylene bisacrylamide bis that react with the free functional groups at the chain termini The pore size of the gel is governed by the concentration of acrylamide and bisacrylamide T and C T concentration of total monomer C proportion of cross linker as a percentage of total monomer The higher the acrylamide concentration
61. ls may be left in SYBR Green I stain for up to 24 hours with little decrease in sensitivity Perform post staining of DNA on TBE or TBE Urea Gels as follows 1 Prepare a 1 10 000 dilution of SYBR Green I reagent in TE 10 mM Tris HCI 1 mM EDTA pH 8 0 TBE or TAE buffer 2 Remove the gel from the cassette using a Gel Knife and place it in a polypropylene staining container 3 Cover the gel with staining solution and incubate at room temperature for 10 40 minutes with gentle agitation Protect the staining container from light by covering it with aluminum foil SYBR Green I stain is compatible with a wide variety of gel reading instruments ranging from ultraviolet transilluminators to argon laser and mercury arc lamp excitation gel scanners SYBR Green I stain is maximally excited at 497 nm but also has secondary excitation peaks at 290 nm and 380 nm The fluorescence emission of SYBR Green I stain bound to DNA is centered at 520 nm 47 Ethidium Bromide Staining Introduction Procedure 48 Note A brief protocol is provided below for staining nucleic acids on TBE and TBE Urea Gels with ethidium bromide Caution Ethidium bromide is a powerful mutagen and is moderately toxic Wear gloves and protective clothing when handling ethidium bromide solutions 1 Prepare 2 pg ml solution of ethidium bromide in ultrapure water 2 Remove the gel from the cassette using a Gel Knife and place it in a staining c
62. m analysis is used for detecting and characterizing Technique metalloproteinases collagenases and other proteases that can utilize casein or gelatin as a substrate Protease samples are denatured in SDS buffer under non reducing conditions and without heating and run on a Zymogram Gel using Tris Glycine SDS Running Buffer After electrophoresis the enzyme is renatured by incubating the gel in Zymogram Renaturing Buffer containing a non ionic detergent The gels are then equilibrated in Zymogram Developing Buffer to add divalent metal cations required for enzymatic activity and then stained and destained Regions of protease activity appear as clear bands against a dark blue background where the protease has digested the substrate Types of Three different types of Zymogram Gels are available from Invitrogen Details Zymogram Gels are listed on the table below Gel Type Separating Gel Substrate Sensitivity Novex Zymogram 10 Tris Glycine gel with 0 1 gelatin 10 units of collagenase Gelatin Gel Novex Zymogram 12 Tris Glycine gel P casein 7 x 10 units of trypsin Casein Gel Novex Zymogram Blue Casein Gel 4 16 Tris Glycine gel blue stained B casein 1 5 x 10 units of trypsin Materials Supplied by the User Important 16 The following reagents are needed to perform electrophoresis with Novex Zymogram Gels Ordering information for pre mixed buffers is on page 63 If you are preparing your own buffers reci
63. ndary structure and amino acid side chains becomes magnified relative to the total size of the peptide Slight differences in protein mobilities also occur when the same proteins are run in different SDS PAGE buffer systems Each SDS PAGE buffer system has a different pH which affects the charge of a protein and its binding capacity for SDS The degree of change in protein mobility is usually small in natural proteins but more pronounced with atypical or chemically modified proteins such as pre stained standards Continued on next page Calibrating Protein Molecular Weight Continued Assigned Apparent Values for apparent molecular weight of Novex molecular weight standards are Molecular Weights derived from the construction of a calibration curve in the Tris Glycine SDS PAGE System We have now calculated and assigned apparent molecular weights for the Novex protein standards in several buffer systems Remember to use the one that matches your gel for the most accurate calibration of your protein The following charts summarize the approximate molecular weight values for the Novex protein molecular weight standards when run in different buffer systems You may generate calibration curves in your lab with any other manufacturer s standards Novex Sharp Pre stained Protein Tris Glycine Gels 4 20 Tricine Gels 10 20 Standard Band 1 260 kDa 260 kDa Band 2 160 kDa 160 kDa Band 3 110 kDa 110 kDa Band 4 80 kD
64. ning Continued Colloidal Blue Staining Kit Protocol A brief staining protocol for staining Novex Gels with the Colloidal Blue Staining Kit is provided below For more details on the staining procedure refer to the Manual IM 6025 This manual is available on our website at www invitrogen com or contact Technical Support see page 76 1 5 Fix the IEF Gel in fixing solution as described on page 21 This step is not required for Tris Glycine Tricine and Zymogram Gels Prepare staining solution for a single gel as described in the table below For two gels double the volume of reagents used for staining Be sure to shake Stainer B prior to making the solution Solutions Tris Glycine Tricine and IEF Gel Zymogram Gel Deionized Water 55 mL 58 mL Methanol 20 mL 20 mL Stainer B 5 mL 2 mL Stainer A 20 mL 20 mL Incubate the gel in this staining solution as follows at room temperature with gentle shaking e Tris Glycine Tricine and Zymogram Gels for a minimum of 3 hours and a maximum of 12 hours e IEF Gels for 30 minutes Decant staining solution and add a minimum of 200 mL of deionized water per gel to the staining container Gently shake gel in water for at least 7 hours Gel will have a clear background after 7 hours in water For gel drying see page 49 Note Novex Gels can be left in deionized water for up to 3 days without significant change in band intensity and background clarity F
65. oiling Anal Biochem 225 351 353 Laemmli U K 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 227 680 685 Ornstein L 1964 Disc Electrophoresis 1 Background and Theory Ann New York Acad Sci 121 321 349 Revzin A 1989 Gel Electrophoresis Assays for DNA Protein Interactions BioTechniques 4 346 355 Schaegger H and von Jagow G 1987 Tricine Sodium dodecyl sulfate Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Range from 1 to 100 kDa Anal Biochem 166 368 379 2010 Life Technologies Corporation All rights reserved The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners Ficoll is a registered trademark of GE Healthcare 78 invitrogen by Lefe technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
66. on 5 mL 10 minutes 10 minutes 10 minutes 10 minutes directly to the gel when the desired staining intensity is reached 8A Decant the Stopping Solution 200mL 10minutes 10 minutes 10 minutes 10 minutes and wash the gel three times 200 mL 10 minutes 10 minutes 10 minutes 10 minutes in ultrapure water 200mL 10minutes 10 minutes 10 minutes 10 minutes 43 SYPRO Ruby Staining Introduction Advantages of SYPRO Ruby Staining Molecular Weight Calibration Materials Supplied by the User 44 Instructions are provided below for a basic and rapid protocol for Novex Pre Cast Gels Novex Tris glycine gels Novex Tricine gels ZOOM gels and Novex IEF gels for the detection of proteins including glycoproteins and phosphoproteins SYPRO Ruby provides the following advantages Linear quantitation range of over three orders of magnitude Compatible with subsequent analysis of proteins by Edman based sequencing or mass spectrometry in 1D or 2D format Compatible with non denaturing gels and IEF gels basic protocol Guidelines and apparent molecular weight values for Novex protein molecular weight standards are provided on page 64 You will need following items for silver staining your gel see page 63 for ordering information on Invitrogen products SYPRO Ruby gel stain Staining containers 1 per gel see below for details Reagent grade methanol Reagent grade glacial acetic acid Trichloroacetic acid for IEF
67. on pages 68 69 e Protein sample e Deionized water e IEF markers e Novex IEF Sample Buffer e Novex IEF Cathode Buffer e Novex IEF Anode Buffer e Fixing solution Prepare 1X IEF Anode Buffer using Novex IEF Anode Buffer 50X 1 Prepare 1 000 mL of IEF Anode Buffer as follows Reagent Amount Novex IEF Anode Buffer 50X 20 mL Deionized Water 980 mL Total Volume 1 000 mL 2 Mix thoroughly Use this buffer to fill the Lower Buffer Chamber of the XCell SureLock Mini Cell for electrophoresis Prepare 1X IEF Cathode Buffer using the appropriate Novex IEF Cathode Buffer pH 3 10 10X or pH 3 7 10X 1 Prepare 200 mL of IEF Cathode Buffer as follows Reagent Amount Novex IEF Cathode Buffer 10X 20 mL Deionized Water 180 mL Total Volume 200 mL 2 Mix thoroughly Use this buffer to fill the Upper Buffer Chamber of the XCell SureLock Mini Cell for electrophoresis Samples for IEF Gels are prepared without SDS to avoid affecting the pl of the protein Reducing agents are also not recommended for the same reason 1 Prepare samples for IEF Gels as described below Reagent Amount Sample x pL Novex IEF Sample Buffer pH 3 10 or pH 3 7 2X 5 pL Deionized Water to 5 uL Total Volume 10 uL 2 Load the sample immediately Do not heat samples for IEF Gels Continued on next page IEF Gels Continued Electrophoresis Conditions Fixing the Gel Staining IEF Gels 2D SDS PAGE with IEF Gels No
68. ontainer 3 Incubate the gel in the ethidium bromide solution for 20 minutes Destain the gel by rinsing the gel three times with ultrapure water for 10 minutes Ethidium bromide staining of polyacrylamide gels requires at least 10 ng of DNA for detection due to the quenching of the fluorescence by polyacrylamide For alternative techniques with greater detection sensitivity perform silver staining using the SilverXpress Silver Staining Kit see page 42 or SYBR Green I staining see page 47 Gel Drying Introduction Materials Supplied by the User DryEase Mini Gel Drying System Dry gels by passive evaporation air drying or vacuum drying Vacuum drying is faster than passive air drying methods but often results in cracked gels due to the speed of dehydration We recommend drying Novex Pre Cast gels using passive air drying methods such as the DryEase Mini Gel Drying System see below For applications that require vacuum drying follow the recommendations on page 51 to minimize cracking of the gels You will need the following items for drying your gel see page 63 for ordering information on Invitrogen products e DryEase Mini Gel Drying System e Gel Dry Drying Solution or prepare your own gel drying solution containing 30 methanol and 5 glycerol e StainEase Gel Staining Tray or a suitable round container Silver stained and Coomassie stained Novex Gels can be dried by vacuum drying or by air
69. ontinued on next page 65 Recipes Continued Tris Glycine SDS Sample Buffer Tris Glycine Native Sample Buffer Tris Glycine Transfer Buffer 66 The Tris Glycine SDS Sample Buffer is available from Invitrogen see page 63 63 mM Tris HCl 10 Glycerol 2 SDS 0 0025 Bromophenol Blue pH 6 8 1 To prepare 10 mL of 2X Tris Glycine SDS Sample Buffer mix the following reagents 0 5 M Tris HCI pH 6 8 2 5mL Glycerol 2mL 10 w v SDS 4mL 0 1 w v Bromophenol Blue 0 5 mL 2 Adjust the volume to 10 mL with ultrapure water Store at 4 C The buffer is stable for 6 months when stored at 4 C The Tris Glycine Native Sample Buffer is available from Invitrogen see page 63 1X composition 100 mM Tris HCl 10 Glycerol 0 0025 Bromophenol Blue pH 8 6 1 To prepare 10 mL of 2X Tris Glycine Native Sample Buffer mix the following reagents 0 5 M Tris HCL pH 8 6 4 mL Glycerol 2mL 0 1 w v Bromophenol Blue 0 5 mL Adjust the volume to 10 mL with ultrapure water Store at 4 C The buffer is stable for 6 months when stored at 4 C The Tris Glycine Transfer Buffer is available from Invitrogen see page 63 12 mM Tris Base 96 mM Glycine pH 8 3 1 To prepare 500 mL of 25 x Tris Glycine Transfer Buffer dissolve the following reagents in 400 mL ultrapure water Tris Base 18 2 g Glycine 90g Mix well and adjust the volume to 500 mL with ultrapure water Store at room temperature The buffer is stable for
70. or long term storage over 3 days keep the gel in a 20 ammonium sulfate solution at 4 C Continued on next page 37 Coomassie Staining Continued Coomassie R 250 Staining Protocol 38 The Coomassie staining protocol described below is recommended for staining Novex Gels You may use any Coomassie staining protocol of choice T Prepare the staining solution containing 0 1 Coomassie R 250 in 40 ethanol 10 acetic acid After electrophoresis incubate 1 or 2 gels in a staining container containing 100 mL Coomassie Blue R 250 staining solution Caution Use caution while performing the following steps using a microwave oven Do not overheat the staining solutions Loosely cover the staining container and heat in a microwave oven at full power for 1 minute To prevent hazardous flammable vapors from forming do not allow the solution to boil Remove the staining container from the microwave oven and gently shake the gel for 15 minutes at room temperature on an orbital shaker Decant the stain and rinse the gel once with deionized water Prepare a destain solution containing 10 ethanol and 7 5 acetic acid Place one or two stained gels in a staining container containing the 100 mL destain solution Loosely cover the staining container and heat in a microwave oven at full power for 1 minute Gently shake the gel at room temperature on an orbital shaker until the desired background is achieved 10 For gel dry
71. pes are provided on pages 65 68 e Protein sample e Deionized water e Protein molecular weight markers e Novex Tris Glycine SDS Sample Buffer e Novex Tris Glycine SDS Running Buffer e Novex Zymogram Renaturing Buffer e Novex Zymogram Developing Buffer e Do not treat zymogram samples with reducing agents Some proteases are multiunit complexes that require the full subunit assembly for activity e Load 2 3 times the recommended amount of unstained molecular weight marker required for a Tris Glycine Gel The marker needs to stain intensely to be visualized against the dark background of the Zymogram Gel e Leave an empty lane between protein molecular weight markers containing reducing agent and protease sample lanes to prevent diffusion of the reducing agent into the protease lane Continued on next page Zymogram Gels Continued Preparing Running Buffer Preparing Samples Electrophoresis Conditions Detecting Protease Activity Preparing Renaturing Buffer Use 1X Novex Tris Glycine SDS Running Buffer for electrophoresis of protease samples on Zymogram Gels 1 Prepare 1 000 mL of Running Buffer as follows Reagent Amount Novex Tris Glycine SDS Running Buffer 10X 100 mL Deionized Water 900 mL Total Volume 1 000 mL 2 Mix thoroughly Use this buffer to fill the Upper and Lower Buffer Chamber of the XCell SureLock Mini Cell for electrophoresis Prepared samples without reducing agents so that
72. phoric acid with 97 6 mL of ultrapure water 2 Store at room temperature The buffer is stable for 6 months when stored at room temperature The TBE Running Buffer is available from Invitrogen see page 63 89 mM Tris base 89 mM Boric acid 2 mM EDTA free acid pH 8 3 1 To prepare 1 000 mL of 5X TBE Running Buffer dissolve the following reagents in 900 mL deionized water Tris Base 54 g Boric acid 2758 EDTA free acid 2 9 g Mix well and adjust the volume to 1 000 mL with ultrapure water Store at room temperature The buffer is stable for 6 months when stored at room temperature 4 For electrophoresis dilute this buffer to 1X with water as described on page 26 The pH of the 1X solution is 8 3 Do not use acid or base to adjust the pH Continued on next page 69 Recipes Continued Hi Density TBE The Hi Density TBE Sample Buffer is available from Invitrogen see page 63 Sample Buffer 18 mM Tris base 18 mM Boric acid 0 4mM EDTA free acid 3 Ficoll Type 400 0 02 Bromophenol Blue 0 02 Xylene Cyanol 1 To prepare 10 mL of 5X Hi Density TBE Sample Buffer dissolve the following reagents in 9 mL deionized water 5X TBE Running Buffer see previous page 2mL Ficoll Type 400 1 5g 1 Bromophenol Blue 1 mL 1 Xylene Cyanol 1mL 2 Mix well and adjust the volume to 10 mL with ultrapure water Store at 4 C The buffer is stable for 6 months when stored at 4 C TBE Urea Sample The TBE Urea Sam
73. ple Buffer is available from Invitrogen see page 63 Buffer 45 mM Tris base 45 mM Boric acid 1 mM EDTA free acid 6 Ficoll Type 400 3 5 M Urea 0 005 Bromophenol Blue 0 025 Xylene Cyanol 1 To prepare 10 mL of 2X TBE Urea Sample Buffer dissolve the following reagents in 9 mL deionized water 5X TBE Running Buffer see previous page 2mL Ficoll Type 400 1 2g 1 Bromophenol Blue 1 mL 1 Xylene Cyanol 0 5 mL Urea 4 2g 2 Mix well and adjust the volume to 10 mL with ultrapure water 3 Store at 4 C The buffer is stable for 3 months when stored at 4 C Continued on next page 70 Recipes Continued Prep TBE Urea Sample Buffer The Prep TBE Urea Sample Buffer is available from Invitrogen see page 63 45 mM Tris base 45 mM Boric acid 1 mM EDTA free acid 6 Ficoll Type 400 3 5 M Urea 1 To prepare 10 mL of 2X Prep TBE Urea Sample Buffer dissolve the following reagents in 9 mL deionized water 5X TBE Running Buffer see page 69 2mL Ficoll Type 400 1 2g Urea 4 2g 2 Mix well and adjust the volume to 10 mL with ultrapure water 3 Store at 4 C The buffer is stable for 6 months when stored at 4 C 71 Gel Migration Charts Novex Tris The migration patterns of protein standards on Novex Tris Glycine Gels are Glycine Gel shown on the table below Use the table to select the proper gel for separating Migration Chart proteins based on size Optimal resolution is achieved when protein
74. presence of SDS leading to various types of gel artifacts If possible change the solubilization agent by dialysis prior to electrophoresis Take the following considerations into account when performing electrophoresis of cell lysates e Genomic DNA in the cell lysate may cause the sample to become viscous and affect protein migration patterns and resolution Shear genomic DNA to reduce viscosity before loading the sample e Cells lysates contain soluble and insoluble fractions The size of each fraction depends upon the type of sample being analyzed The nature of the insoluble fraction may result in altered protein migration patterns and resolution Separate the two fractions by centrifugation and load them on separate lanes for electrophoresis e If RIPA buffer is used in cell lysis subsequent blotting of proteins lt 40 kDa may be inhibited due to the presence of Triton X 100 in the buffer 11 Tris Glycine Gels Tris Glycine Discontinuous Buffer System Materials Supplied by the User Preparing Running Buffer 12 Novex Tris Glycine gels are based on the Laemmli System Laemmli 1970 with minor modifications for maximum performance in the pre cast format Unlike traditional Laemmli gels with a stacking gel pH of 6 8 and separating gel pH of 8 8 Novex Tris Glycine gels have a pH of 8 65 for both regions The Tris Glycine discontinuous buffer systems utilizes three ions e Chloride from the gel buffer serves
75. roducts e Staining container e Deionized water e Orbital Shaker For SimplyBlue SafeStain see page 36 e SimplyBlue SafeStain e Optional 20 NaCl e Optional Microwave oven e 12 Trichloroacetic acid for IEF gels For Colloidal Blue Staining Kit see page 37 e Colloidal Blue Staining Kit e Methanol e Optional 20 Ammonium sulfate e Fixing solutino for IEF gels For Coomassie R 250 Staining see page 38 e 0 1 Coomassie R 250 in 40 ethanol and 10 acetic acid e Destaining Solution consisting of 10 ethanol and 7 5 acetic acid e Optional Microwave oven Continued on next page 35 Coomassie Staining Continued SimplyBlue SafeStain Protocol 36 TM The Basic Protocol for staining Novex Gels with SimplyBlue SafeStain is provided below For the Microwave Protocol and staining large format gels refer to the SimplyBlue SafeStain Manual IM 6050 This manual is available on our website at www invitrogen com or contact Technical Support page 76 For general use with 1 0 mm and 1 5 mm thick Tris Glycine Gels and 1 0 mm thick Tricine Zymogram and IEF Gels 8 cm x 8 cm After electrophoresis follow the instructions below Be sure the mini gel moves freely in water or stain to facilitate diffusion during all steps Note Stain Zymogram Gels with SimplyBlue SafeStain after renaturing and developing the gel for enzyme activity 1 Fix IEF Gels in 100 mL 12 TCA for 15 minutes Th
76. rogen products e Blotting membranes e Filter paper not needed if using Novex pre cut membrane filter paper sandwiches e Methanol if using PVDF membranes e Appropriate Transfer Buffer e Deionized water For blotting Tris Glycine Tricine and IEF Gels use 1X Tris Glycine Transfer Buffer If you are preparing your own transfer buffer see page 66 for a recipe An alternate transfer protocol for IEF Gels is provided on page 57 If you are performing protein sequencing an alternate transfer buffer compatible with the technique is listed on the next page Prepare 1 000 mL of Transfer Buffer Tris Glycine Transfer Buffer 25X 40 mL Methanol 200 mL Deionized Water 760 mL Total Volume 1 000 mL Continued on next page Blotting Novex Pre Cast Gels Continued Preparing Transfer Buffer for TBE Gels Preparing Transfer Buffer Compatible with Protein Sequencing Preparing Blotting Pads Preparing Transfer Membrane and Filter Paper For blotting TBE and TBE Urea Gels use 0 5X TBE Running Buffer If you are preparing your own transfer buffer see page 69 for a recipe Prepare 1 000 mL of 1X Tris Glycine Transfer Buffer using the Tris Glycine Transfer Buffer 25X as follows TBE Running Buffer 5X 40 mL Methanol 200 mL Deionized Water 760 mL Total Volume 1 000 mL For blotting TBE and TBE Urea Gels Dilute the 5X TBE Running Buffer to 0 5X with deionized water Tris Glycine Transfer Buffer interferes with
77. rophoresis do not have a net charge which may cause problems during the transfer Some native proteins may have a higher pI than the pH of the Tris Glycine Transfer Buffer used in standard transfer protocols Guidelines are provided below to increase the transfer efficiency of native proteins e Increasing the pH of the transfer buffer to 9 2 25 mM Tris Base 25 mM glycine pH 9 2 allows proteins with pI below 9 2 to transfer towards the anode electrode e Place a membrane on both sides of the gel if you are using the regular Tris Glycine Transfer Buffer pH 8 3 If there are any proteins that are more basic than the pH of the transfer buffer they will be captured on the membrane placed on the cathode side of the gel e Incubate the gel in 0 1 SDS for 15 minutes before blotting with Tris Glycine Transfer Buffer The small amount of SDS will render enough charge to the proteins so they can move unidirectionally towards the anode and in most cases will not denature the protein Native proteins may diffuse out of the membrane into the solution during the blocking or antibody incubation steps as the native proteins tend to be more soluble To prevent diffusion of the proteins out of the membrane we recommend fixing the proteins to the membrane by air drying the membrane or incubating the membrane in 5 10 acetic acid for 15 minutes followed by rinsing the membrane with deionized water and then air drying By performing any of these two fixing
78. roteins using SDS PAGE Proteins are separated based on their molecular weight using denaturing polyacrylamide gel electrophoresis The gel is stained after 2D electrophoresis to visualize the separated proteins or the proteins are blotted onto membranes Protein spots can be excised from the gel or membranes and subjected to further analyses such as mass spectrometry or chemical microsequencing to facilitate protein identification During IEF proteins migrate in an electric field until a stable pH gradient is formed and the proteins settle into their pI A high finishing voltage is applied to focus the proteins into narrow zones High voltage cannot be used during the initial stages of IEF as movement of carrier ampholytes generate excessive heat To obtain the best results IEF is typically performed by gradually increasing the voltage then maintaining the final focusing voltage for 30 minutes Alternatively IEF can be performed at constant power so the voltage will increase as the current decreases Continued on next page 19 IEF Gels Continued Materials Supplied by the User Preparing Anode Running Buffer Lower Buffer Chamber Preparing Cathode Running Buffer Upper Buffer Chamber Preparing Sample 20 The following reagents are needed to perform isoelectric focusing with Novex IEF Gels Ordering information for pre mixed buffers is on page 63 If you are preparing your own buffers recipes are provided
79. rritants commonly used in Blot Module electrotransfer Transferring One Gel 1 10 11 12 54 After opening the gel cassette as described on page 34 remove wells with the Gel Knife Place a piece of pre soaked filter paper on top of the gel with the edge above the slot in the bottom of the cassette leaving the foot of the gel uncovered Keep the filter paper saturated with the transfer buffer and remove all trapped air bubbles by gently rolling over the surface using a glass pipette as a roller Turn the plate over so the gel and filter paper are facing downwards over a gloved hand or clean flat surface Use the Gel Knife to push the foot out of the slot in the plate and separate the gel from the plate When the gel is on a flat surface cut the foot off the gel with the Gel Knife Wet the surface of the gel with transfer buffer and position the pre soaked transfer membrane on the gel ensuring all air bubbles have been removed Place another pre soaked filter paper on top of the membrane Remove any trapped air bubbles Place two soaked blotting pads into the cathode core of the blot module The cathode core is the deeper of the two cores and the corresponding electrode plate is a darker shade of gray Carefully pick up the gel membrane assembly and place it on the pad such that the gel is closest to the cathode plate see Figure 1 next page Add enough pre soaked blotting pads to raise the assembly 0 5
80. s Refer to pages 39 47 for more information on these techniques 27 TBE Urea Gels Introduction Materials Supplied by the User Preparing Running Buffer 28 Novex denaturing polyacrylamide TBE Urea Gels provide high resolution of short single strand oligonucleotides The gels provide excellent resolution for fast size and purity confirmations of DNA or RNA oligos from 20 600 bases The TBE Urea Gels contain 7 M urea for maximum denaturation The following reagents are needed to perform gel electrophoresis with Novex TBE Urea Gels Ordering information for pre mixed buffers is on page 63 If you are preparing your own buffers recipes are provided on pages 69 70 e DNA or RNA sample e Deionized water e Appropriate DNA or RNA markers e Novex TBE Urea Sample Buffer e Novex Prep TBE Urea Sample Buffer for preparative electrophoresis only e Novex TBE Running Buffer To obtain optimal results with TBE Urea Gels observe the following recommendations e Use RNase free ultrapure water e Prior to loading samples flush wells several times with 1X TBE Running Buffer to remove urea e Load samples quickly and avoid allowing the gel to stand for long periods of time after loading to prevent diffusion e Use Prep TBE Urea Sample Buffer for preparative gel electrophoresis as this buffer does not contain any marker dyes e Wear gloves and use dedicated equipment to prevent contamination e Avoid using buffers with
81. s conditions Staining the Gel Gel shift assays use labeled radioactive fluorescent biotin DNA fragments for visualization of results Use the appropriate technique to develop the image for the type of label you are using 31 Electrophoresis of Novex Pre Cast Gels Introduction Instructions are provided below for electrophoresis of Novex Pre Cast Gels using the XCell SureLock Mini Cell For more information on the XCell SureLock Mini Cell refer to the manual IM 9003 This manual is available on our website at www invitrogen com or contact Technical Support see page 76 For information on sample and buffer preparation for Novex Pre Cast Gels see pages 10 31 Protocol using Wear gloves and safety glasses when handling gels XCell SureLock XCell SureLock Mini Cell requires 200 mL for the Upper Buffer Chamber and Mini Cell 600 mL for the Lower Buffer Chamber 1 Remove the Novex Pre Cast Gel from the pouch 2 Rinse the gel cassette with deionized water Peel off the tape from the bottom of the cassette 3 Gently pull the comb out of the cassette in one smooth motion Rinse the sample wells with the appropriate 1X Running Buffer Invert the gel and shake the gel to remove the buffer Repeat two more times 5 Orient the two gels in the Mini Cell such that the notched well side of the cassette faces inwards toward the Buffer Core Seat the gels on the bottom of the Mini Cell and lock into place with the
82. s conditions The size of the single strand DNA fragments visualized at the dye fronts of the different TBE Urea Gels is shown in the table below Gel Type Dye Front Bromophenol Blue Xylene Cyanol light blue dark blue 6 TBE Urea Gel 25 bases 110 bases 10 TBE Urea Gel 20 bases 55 bases 15 TBE Urea Gel 10 bases 40 bases accuracy is 5 bases Novex TBE Urea Gels can be stained by silver staining ethidium bromide and SYBR Green staining techniques after electrophoresis Refer to pages 35 48 for more information on these techniques 29 DNA Retardation Gels Gel Shift Assay Materials Supplied by the User Preparing Samples 30 Note Novex DNA Retardation Gels consist of 6 polyacrylamide prepared with 0 5X TBE as the gel buffer The 6 gel provides good resolution of fragments in the range of 60 2500 bp used for DNA retardation assays The gel shift assay is based on the fact that the movement of a DNA molecule through a non denaturing polyacrylamide gel is hindered when bound to a protein molecule Revzin 1989 This technique is used to characterize DNA protein complexes The 0 5X TBE buffer is sufficient for good electrophoretic separation yet low enough to promote DNA protein interactions Detection is performed with ethidium bromide staining of DNA or for greater sensitivity with radiolabeling the DNA or protein The following reagents are needed to perform gel electrophoresis with Novex
83. s per unit distance The higher the voltage the faster an ion moves For most applications we recommend a constant voltage setting e A constant voltage setting allows the current and power to decrease over the course of electrophoresis providing a safety margin in case of a break in the system e The constant voltage setting does not need adjustment to account for differences in number or thickness of gels being electrophoresed Current For a given gel buffer system at a given temperature current varies in proportion to the field strength voltage and or cross sectional area thickness and or number of gels When using a constant current setting migration starts slow and accelerates over time thus favoring stacking in discontinuous gels When running under constant current set a voltage limit on the power supply at or slightly above the maximum expected voltage to avoid unsafe conditions At constant current voltage increases as resistance increases If a local fault condition occurs e g a bad connection high local resistance may cause the voltage to reach the maximum for the power supply leading to overheating and damage of the electrophoresis cell Power Wattage measures the rate of energy conversion which is manifest as heat generated by the system Using constant power ensures that the total amount of heat generated by the system remains constant throughout the run but results in variable mobility since voltage incre
84. sessesansersunensersenense 33 Opening Novex Pre Cast Gel Cassettes uni 34 COOMASSIE StaIMING o eere aaee a aa aaea eaae E re aee Aaa Taa EAE TAEL Tea A Re RS 35 A ni aaae aaa e E R R a A a dats R ES 39 SYPROY R Staini gii ei e is e A ses 44 OD RE Green Staining A o 47 Ethidium Bromide Staining soenen ina ae eroare Ee e Eaa Eea aaa ESE Raoa n TEn n Reena eiA 48 Cory iia a E S A E ES 49 Blotting Novex Pre Cast rl a 52 Calibrating Protein Molecular Weight cccccccsesesssseessseececeeeceneesesesesnsneneneseseseseeseceeesenenesesesnananenes 58 Troubleshooting e a eae A ete CET eE aa aa TO Lah east K ii 61 iii APpendiX iaiia isinira daei aaisa iaeiaiai aiia e aa Naia Sidana 63 ACCOSSOFY Products lala Midas S ata tanh E E iia no 63 REP ta te a e led eLo 65 GellMigration Charts martirio iaa ias 72 Technical Support titi iaa e a e E a a odes ikon Seuss Lida ade EO Oa eA Eo AAE divisas 76 References tata aae ies a EA EE EE ana 78 General Information Purpose of the Guide Storage and Shelf life Packaging Handling the Gels Intended Use TM A variety of pre cast gels for use with the XCell SureLock Mini Cell are available from Invitrogen These include gels for analysis of proteins Tris Glycine Tricine Zymogram IEF and ZOOM Gels and nucleic acids TBE TBE Urea and DNA Retardation The Novex Pre Cast Gel Electrophoresis Guide contains information about the Novex Pre Cast gels and is intended to
85. sults follow these guidelines Be sure to wear clean gloves that have been rinsed with deionized water while handling gels Use clean containers and designate these containers for silver staining purposes only Make sure the size of the container permits free movement of the gel during shaking and complete immersion in solution while staining Do not touch the gel with bare hands or metal objects and do not put pressure on gels while handling or changing solutions Use teflon coated stir bars and clean glass containers to prepare reagents Avoid cross contamination of kit reagents Use freshly made solutions Use the reagents provided in the SilverQuest Silver Staining Kit to prepare the following solutions for staining Sensitizing solution Ethanol 30 mL Sensitizer 10 mL Ultrapure water to 100 mL Staining solution Stainer 1mL Ultrapure water to 100 mL Developing solution Developer 10 mL Developer enhancer 1 drop Ultrapure water to 100 mL Note You may prepare all solutions immediately before starting the staining protocol or prepare them as you proceed to the next step Continued on next page Silver Staining Continued SilverQuest Microwave Silver Staining Protocol The Fast Staining protocol using a microwave oven for silver staining Novex Gels using SilverQuest Silver Staining Kit is described below For the Basic Protocol and more details on the staining procedure refer to the SilverQuest Silver Stain
86. t analysis Methods of transfer include wet semi wet semi dry and dry blotting Semi dry blotting can be performed with the Novex Semi Dry Blotter and dry blotting is performed with the iBlot Gel Transfer Device Refer to the respective manuals for information on blotting with these devices Instructions are provided below for semi wet blotting of Novex Pre Cast Gels using the XCell II Blot Module For more information on the XCell IT Blot Module refer to the manual IM 9051 available at www invitrogen com or contact Technical Support see page 76 If you are using any other blotting apparatus follow the manufacturer s recommendations During blotting the distance traveled gel thickness between the electrodes is much lower than during electrophoresis requiring lower voltage and lower field strength volts distance However the cross sectional area of current flow is much greater requiring higher current Blotting power requirements depend on field strength electrode size and conductivity of transfer buffer The higher the field strength and conductivity of the buffer the higher is the current requirement the current decreases during the run as the ions in the buffer polarize It is important to use a power supply capable of accommodating the initial high current requirement In addition to the XCell II Blot Module the following reagents are needed for blotting your gel see page 63 for ordering information on Invit
87. te See page 32 for instructions on running Novex Pre Cast Gels using the XCell SureLock Mini Cell Run the gel at 100 V constant for 1 hour followed by 200 V constant for 1 hour and finish with 500 V constant for 30 minutes See page 33 for additional details on electrophoresis conditions Fixing the proteins in the IEF gel is recommended after electrophoresis The fixing step also helps to remove carrier ampholytes from the gel resulting in lower background after staining Fixing solution consists of 12 TCA or 12 TCA wtih 3 5 sulfosalicylic acid 1 Prepare 500 mL of fixing solution as follows Reagent Amount Trichloroacetic Acid TCA 60 0 g Sulfosalicylic Acid optional 17 5 g Deionized Water to 500 mL Total Volume 500 mL Mix solution thoroughly Fix gels for 30 minutes IEF gels can be stained by Coomassie or colloidal blue techniques refer to pages 35 38 TM If using the SimplyBlue SafeStain wash the gel extensively to remove traces of TCA from the fixation process to avoid formation of precipitate in the gel After staining the gel and documenting the results proteins separated by pI can be separated by mass We recommend using NuPAGE Bis Tris or Novex Tris Glycine Gels with a 2D well or ZOOM Gels for 2D SDS PAGE 2D wells can fit strips of 6 5 cm while ZOOM IPG wells can fit strips of 7 cm Fixing and staining the IEF gel prior to performing second dimension SDS PAGE has the follow
88. the Lower Buffer Chamber with deionized water by pouring approximately 650 mL in the gap between the front of the blot module and the front of the Lower Buffer Chamber The water level should reach approximately 2 cm from the top of the Lower Buffer Chamber This serves to dissipate heat produced during the run 15 Place the lid on top of the unit 16 With the power turned off plug the red and black leads into the power supply Refer to Recommended Transfer Conditions on the next page for transfer conditions Transferring Two Gels in One Blot Module 1 Repeat Steps 1 7 previous page twice to make two gel membrane assemblies 2 Place two pre soaked pads on cathode shell of blot module Place the first gel membrane assembly on the pads such that the gel faces the cathode plate See Figure 2 3 Add another pre soaked blotting pad on top of first gel membrane assembly 4 Position second gel membrane assembly on top of blotting pad with the gel facing the cathode side 5 Proceed with steps 8 13 from Transferring One Gel 6 Refer to Recommended Transfer Conditions on the next page for transfer conditions Figure 1 Figure 2 0d Blotting Pad e 4 Blotting Pad laa a AAA Blotting Pad I POS FSA Blotting Pad Filter Paper l l Filter Paper EE Transfer Membrane l _ gt gt gt Transfer Membrane 1 S Gel l OOGG E EA E E Second Gel IO Filter Paper Filter Paper Cee a OSA AA AA Blotting Pad I ie ee
89. the step one more time 6 Place the gel in 100 mL of Staining solution Microwave at high power for 30 seconds Remove the gel from the microwave and gently agitate it for 5 minutes at room temperature 7 Decant the Staining solution and wash the gel with 100 mL of ultrapure water for 20 60 seconds Do not wash the gel for more than a minute 8 Place the gel in 100 mL of Developing solution and incubate for 5 minutes at room temperature with gentle agitation on a rotary shaker Do not microwave 9 Once the desired band intensity is achieved immediately add 10 mL of Stopper directly to the gel still immersed in Developing solution and gently agitate the gel for 10 minutes The color changes from pink to clear indicating the end of development 10 Wash the gel with 100 mL of ultrapure water for 10 minutes For gel drying see page 49 If you need to destain the gel for mass spectrometry analysis see the SilverQuest Silver Staining Kit Manual IM 6070 Continued on next page 41 Silver Staining Continued Preparing Solutions for SilverXpress Silver Staining 42 Prepare the reagents as described below If you are staining two gels double the reagent volumes Fixing solution for Tris Glycine and Tricine Gels Methanol 100 mL Acetic Acid 20 mL Ultrapure water to 200 mL Fixing solution for TBE TBE Urea Gels Sulphosalicylic acid 78 TCA 24g Ultrapure water to 200 mL Sensitizing solution Methanol 100 mL Sensit
90. uffer system gel format and thickness for your application Review the following section and Well Volume page 8 to determine the type of gel that is best suited for your application Refer to the Novex Gel Migration Charts see page 72 to find the gel with the region of maximum resolution best suited for your sample The leading protein molecules should migrate about 70 of the length of gel for best resolution Separation of proteins over a wide range of molecular weights Use Novex Tris Glycine Gels for separating proteins over a wide molecular weight range 6 200 kDa under denaturing or non denaturing conditions Resolve large molecules with low percentage gels and small molecules with high percentage gels If the molecular weight of the molecule is unknown or the sample contains a wide range of molecules use a gradient gel Separation of low molecular weight proteins and peptides The Novex Tricine Gels provide high resolution of low molecular weight proteins and peptides 2 200 kDa Tricine gels give the best results under denaturing conditions Isoelectric focusing IEF Use Novex IEF Gels for native vertical IEF of proteins The pH 3 10 gels have a pI performance range of 3 5 8 5 and pH 3 7 gels have a pI performance range of 3 0 7 0 Protease detection The Novex Zymogram Gels are used for detecting and characterizing proteases that utilize casein or gelatin as the substrate Proteins are run under denat
91. uring conditions and then renatured for enzymatic activity 2D separation of proteins The ZOOM Gels are specifically designed for second dimension electrophoresis of 7 0 cm IPG strips Gels with 2D wells can also be used but only accommodate IPG strips of 6 5 cm Continued on next page Gel Selection Continued Nucleic Acid Separation Applications Nucleic acid analysis The Novex Pre Cast Gels are capable of resolving nucleic acids in the range of 10 3000 bp Novex TBE Gels are used to perform analysis of DNA fragments from restriction digest and PCR products Southern analysis and primer analysis Novex TBE Urea Gels are used for denaturing nucleic acid analysis and are suited for RNase Protection Assays in vitro transcription studies RNA stability studies and oligonucleotide purification Gel shift assays The Novex 6 DNA Retardation Gels are used to perform gel shift assays Well Volume Recommended Loading Volumes The recommended loading volumes and protein load per band by the detection method are provided in the table below Note The 9 well gels are compatible with any eight channel pipettors used for loading samples from 96 well plates An additional lane is included for loading protein molecular weight standard Well Types Maximum Load Maximum Protein Load Per Band by Detection Method Volume Coomassie Staining Ethidium Bromide Silver Staining 1 0mm 700 pL 12 pg band 2 4 ug band Sc
92. uring native electrophoresis proteins are separated based on their charge to mass ratios Reducing Electrophoresis is performed under reducing conditions using reducing agents such as dithiothreitol DTT or B mercaptoethanol B ME The reducing agents cleave any disulfide bonds between cysteine residues resulting in complete separation of denatured proteins into their individual subunits Continued on next page Overview of Electrophoresis Continued Power Supply Considerations for Electrophoresis In electrical terms the process of electrophoresis is closely associated with the following equations derived from Ohm s Law Voltage Current x Resistance V IR Wattage Current x Voltage W IV Resistance The electrical resistance of the assembled electrophoresis cell is dependent on buffer conductivity gel thickness temperature and the number of gels being run Although the resistance is determined by the gel system the resistance varies over the course of the run e In discontinuous buffer systems and to a lesser extent in continuous buffer systems resistance increases over the course of electrophoresis This occurs in the Tris Glycine buffer system as highly conductive chloride ions in the gel are replaced by less conductive glycine ions from the running buffer e Resistance decreases as the temperature increases Voltage The velocity of an ion in an electric field varies in proportion to the field strength Volt
93. vex Bis Tris ZOOM Gels or at 125 V for 90 minutes for Novex Tris Glycine ZOOM Gels Stain the gel with the appropriate method for the type of gel and sample amount after electrophoresis Refer to the techniques described on pages 35 46 25 TBE Gels Introduction Advantages of TBE Gels Materials Supplied by the User Preparing Running Buffer 26 Novex polyacrylamide TBE Gels provide high resolution analysis of restriction digests and PCR products The TBE Gels give sharp intense bands and provide separations of double strand DNA fragments from 10 3 000 base pairs Using polyacrylamide gels for nucleic acid separation provides the following advantages over agarose gels High resolution and sensitivity Lower background staining Requires less sample concentration and volume Efficient blotting Easy to extract DNA from the gel and does not interfere with enzymatic reactions Accurate and reproducible results The following reagents are needed to perform gel electrophoresis with Novex TBE Gels Ordering information for pre mixed buffers is on page 63 If you are preparing your own buffers recipes are provided on pages 69 70 DNA sample Deionized water Appropriate DNA markers Novex Hi Density TBE Sample Buffer Novex TBE Running Buffer Use 1X Novex TBE Running Buffer to perform electrophoresis 1 Prepare 1 000 mL of Running Buffer as follows Reagent Amount Novex TBE Running Buffer
94. x Tris Glycine SDS or 10X Native Running Buffer 100 mL Deionized Water 900 mL Total Volume 1 000 mL 2 Mix the buffer thoroughly and use it to fill the Upper and Lower Buffer Chambers of the assembled XCell SureLock Mini Cell for electrophoresis Continued on next page Tris Glycine Gels Continued Preparing Samples for Denaturing Electrophoresis Preparing Samples for Native Electrophoresis Electrophoresis Conditions Staining the Gel To separate proteins by mass alone denature samples using SDS Sample Buffer and heating 1 Prepare each sample as described below Reagent Amount Sample x pL Novex Tris Glycine SDS Sample Buffer 2X 5 uL Deionized Water to 5 uL Total Volume 10 pL To separate proteins by charge mass ratio in their native conformation use Heat the sample at 85 C for 2 minutes Load the samples onto the gel immediately Note For reduced samples add the reducing agent to a final concentration of 1X immediately prior to electrophoresis to obtain the best results non denaturing native electrophoresis 1 Prepare each sample as described below Reagent Amount Sample x pL Novex Tris Glycine Native Sample Buffer 2X 5 pL Deionized Water to 5 uL Total Volume 10 pL Load the samples onto the gel immediately Do not heat samples for native electrophoresis See page 32 for instructions on running Novex Pre Cast Gels using the XCell TM SureLock Mini Cell Run th

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Novex® Pre-Cast Gel Electrophoresis Guide