XCell II™ Blot Module - Thermo Fisher Scientific
Contents
1. Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen 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 Invitrogen assumes 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 2009 Life Technologies Corporation All rights reserved Parafilm is a registered trademark of American National Can Coomassie is a registered trademark of Imperial Chemical Industries invitrogen Corporate Headquarters 5791 Van Allen Carlsb T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com2. 2 After electrophoresis of the gel equilibrate the gel in 0 7 acetic acid for 10 minutes Tip The 5 polyacrylamide gels are more sticky and difficult to handle than higher percentage polyacrylamide gels To lift the gel from the equilibration solution submerge the filter paper under the gel while the gel is floating in the equilibration solution When the gel is in the correct position lift up the filter paper to attach the gel to the filter paper This prevents the gel from sticking to the filter paper before it is in the proper position and avoids handling of the gel 3 Assemble the gel membrane sandwich as described on page 12 except in a reverse order so that the membrane is on the cathode side of the gel This is the opposite of a typical western blotting protocol where the negatively charged protein will migrate toward the anode during the transfer 4 Transfer for 1 hour at 10 V constant Continued on next page 27 Special Applications Continued Blotting During SDS PAGE all proteins have a net negative charge Native Gels due to the SDS in the sample buffer and the running buffer Proteins separated during native gel electrophoresis do not have a net charge which may cause problems during the transfer It is possible that some native proteins may have a higher pI than the pH of the Tris Glycine Transfer Buffer used for standard western transfer protocols Review the guidelines below to increase the transfer e
3. Nylon Membrane 0 45 um pore size WesternBreeze Chromogenic Western Blot Detection Kit WesternBreeze Chemiluminescent Western Blot Detection Kit Novex Tris Glycine Transfer Buffer 25X NuPAGE Transfer Buffer 20X Novex TBE Running Buffer 5X NuPAGE Antioxidant 32 Quantity 10 gels box 10 gels box 1 unit Each Each 1 unit 2 x 250 uL 250 uL 1L 20 membrane filter paper sandwiches 20 membrane filter paper sandwiches 20 membrane filter paper sandwiches 20 membrane filter paper sandwiches 1 Kit Anti Mouse 1 Kit Anti Rabbit 1 Kit Anti Mouse 1 Kit Anti Rabbit 500 mL 125 mL 1L 15 mL Catalog no NP0321BOX EC6075BOX EI0002 E19052 E19016 E18600 LC5800 LC5602 LC6060 LC2000 LC2002 LC2005 LC2003 WB7103 WB7105 WB7104 WB7106 LC3675 NP0006 LC6675 NP0005 Technical Support World Wide Visit the Invitrogen website at www invitrogen com for Web i 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 Additional product information and special offers Contact us For more information or technical assistance call write fax or email Additional international offices are listed on our web page www invitrogen com Corporate Headquarters European Headquarters 5791
4. Follow local municipal waste ordinances for proper disposal provisions to reduce the environmental impact of WEEE Visit www invitrogen com weee for collection and recycling options Overview Introduction Advantages of Blotting XCell II Blot Module Western or immuno blotting is the transfer of separated proteins in a gel to the surface of a thin support membrane matrix The proteins are bound and immobilized on the membrane Southern and northern blotting involves the transfer of separated DNA and RNA respectively from a gel to a membrane To perform a transfer the gel is layered next to a membrane and placed in a voltage gradient perpendicular to the gel Negatively charged molecules will migrate out from the gel move towards the positive electrode and get deposited on the membrane The advantages of blotting include e Removal of gel impurities from the protein or nucleic acid which may hinder further analysis e Easy access of proteins or nucleic acids on a blot for further analysis e Faster analysis of proteins on the blots as protein diffusion is minimized e Shorter staining and destaining time of proteins on the blot as compared to gels e Multiple successive reprobing of a single blot for analyses after removal of the probe each time e Convenient way to store separated proteins or nucleic acids for future use The XCell II Blot Module is a simple apparatus designed for blotting of mini gels and is
5. module correctly to prevent any the buffer volume in the leaking module Power supply High ionic strength of the Prepare the buffer as described shuts off using transfer buffer on page 8 a Power supply is Use a power supply with higher ant operating at a current limits conditions ek close to the current limit of the power supply Continued on next page 24 Troubleshooting Continued Diffuse bands Poor contact between the Roll over the surface of each and swirling gel and the membrane layer of the gel membrane pattern on the sandwich with a glass pipette to membrane ensure good contact between the gel and the membrane Saturate the blotting pads with transfer buffer to remove air bubbles Under or Add or remove blotting pads to overcompression of the prevent any type of compression gel of the gel Empty spots on Presence of air bubbles Be sure to remove all air bubbles the membrane between the gel and the between the gel and membrane membrane preventing the by rolling a glass pipette over transfer of proteins the membrane surface Expired or creased Use fresh undamaged membranes used membranes Poor transfer Membrane not treated Be sure that the membrane is efficiency with properly before use pre wetted with methanol or PVDF ethanol Poor contact between the Use more blotting pads or membrane and the gel replace the old blotting pads with new ones Overcompression of the Remove enough blotting
6. Van Allen Way Inchinnan Business Park Carlsbad CA 92008 USA 3 Fountain Drive Tel 1 760 603 7200 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Tech Fax 44 0 141 814 6117 E mail E mail tech_support invitrogen com eurotech invitrogen com SDS Safety Data Sheets SDSs are available on our website at Requests www invitrogen com sds Continued on next page 33 Technical Support Continued Limited Warranty 34 Invitrogen 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 Invitrogen warrants that all of its products will perform according to the 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 Invitrogen Corporation s liability only to the cost 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 Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order
7. pads so gel indicated by a that the unit can be closed flattened gel without exerting pressure on the gel and the membrane High Insufficient blocking of Increase the blocker background on non specific sites concentration or the incubation western blots time 25 Special Applications Introduction Materials Needed 26 Additional protocols are provided below for certain special transfer applications such as blotting of IEF gels next page native gels page 28 and semi dry blotting page 29 using the XCell II Blot Module These protocols are slightly different than the standard blotting protocol described on page 12 e Previously electrophoresed mini gels maximum gel size 9 cm x 9 cm e 0 7 acetic acid in deionized water for blotting IEF gels e Native transfer buffer for blotting native gels 25 mM Tris Base 25 mM glycine pH 9 2 For semi dry blotting e Methanol Semi dry transfer unit Filter papers NuPAGE Transfer Buffer NuPAGE Antioxidant Continued on next page Special Applications Continued Blotting IEF Gels Novex pre cast 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 is optimized to prevent hydrolysis and effective transfer of basic proteins due to the low pH of the transfer buffer 1 Chill the 0 7 acetic acid which will be used later for transfer
8. the next page Staining the membrane Stain the membrane after the transfer to evaluate the transfer efficiency If you are using the membrane for peptide sequencing you may stain the membrane with Coomassie Blue stain If you are using the membrane for immunodetection you can stain the membrane with a temporary stain such as Ponceau S see page 18 for Ponceau S staining After visualizing the transferred protein bands on the membrane you can rinse the membrane with deionized water to completely remove the staining or incubate the membrane directly in the blocking solution Ponceau S stain does not interfere with most immunodetection methods Optimizing Blotting Parameters Introduction Gel Percentage Gel Thickness Field Strength Alcohol in Transfer Buffer Each parameter of the blotting protocol plays a role during the transfer of proteins An ideal blotting protocol balances each parameter to provide efficient transfer of proteins When using the XCell IT Blot module most proteins will transfer efficiently using the protocol on page 12 Based on specific properties of a protein or a set of proteins some optimization of the blotting protocol may be necessary Review the points described below to optimize the blotting protocol Choose the lowest percentage acrylamide appropriate for the molecular weight of your protein or proteins of interest Gradient gels are excellent for blotting a range of protein size
9. to avoid exposure to irritants commonly used in electrophoresis and blotting procedures Do not touch the membrane or gel with bare hands This may contaminate the gel or membrane and interfere with further analysis Prepare the appropriate buffer for your gel type from the recipes given below For Blotting Novex Tris Glycine or Tricine Gels Prepare 1 000 mL of 1X Novex Tris Glycine Transfer Buffer using Novex Tris Glycine Transfer Buffer 25X as follows Novex Tris Glycine Transfer Buffer 25X 40 mL Methanol 200 mL Deionized Water 760 mL Total Volume 1 000 mL See 30 for a recipe of Tris Glycine Transfer Buffer if you are preparing your own transfer buffer Continued on next page Preparing for Transfer Continued Preparing Transfer Buffer continued Preparing Blotting Pads For Blotting NuPAGE Novex Bis Tris or Tris Acetate Gels Prepare 1 000 mL of 1X NuPAGE Transfer Buffer using the NuPAGE Transfer Buffer 20X as follows Reagents Reduced Non Reduced NuPAGE Transfer Buffer 20X 50 mL 50 mL NuPAGE Antioxidant 1 mL Methanol 100 mL 100 mL Deionized Water 849 mL 850 mL Total Volume 1 000 mL 1 000 mL NuPAGE Transfer Buffer with 10 methanol provides optimal transfer of a single gel in the blot module If you are transferring 2 gels in the blot module increase the methanol content to 20 to ensure efficient transfer of both gels Refer to page 30 for a recipe of the NuPAG
10. E Transfer Buffer if you are preparing your own transfer buffer For Blotting Novex TBE or DNA Retardation Gels Prepare 1 000 mL of 0 5X TBE transfer buffer using the Novex TBE Running Buffer 5X as follows Novex TBE Running Buffer 5X 100 mL Deionized Water 900 mL Total Volume 1 000 mL See page 30 for a recipe of the Novex TBE Running Buffer if you are preparing your own transfer buffer Use 700 mL of transfer buffer to soak the blotting pads until saturated Remove air bubbles by squeezing the blotting pads while they are submerged in buffer Removing air bubbles is essential as they can block the transfer of biomolecules Continued on next page Preparing for Transfer Continued Preparing Use Novex pre cut membrane filter paper sandwiches see Transfer page 32 for ordering information or cut selected transfer Membrane membrane and filter paper to the dimensions of the gel and Filter e PVDF membrane Pre wet the PVDF membrane for Paper 30 seconds in methanol ethanol or isopropanol Briefly rinse in deionized water and then place the membrane in a shallow dish containing 50 100 mL transfer buffer for several minutes e Nitrocellulose Nylon membrane Place the membrane directly in a tray containing the transfer buffer for several minutes e Filter paper Soak briefly in transfer buffer immediately before using e Gel Use the gel immediately following the run page 11 Do not soak the gel in trans
11. andards page 32 Needed Ponceau S or Coomassie stain Transfer membrane Previously transferred mini gel maximum gel size 9 cm x 9 cm Continued on next page 19 Testing the Efficiency of Blotting Continued Procedure 20 The following methods can be used to test the efficiency of protein transfer Using pre stained standards This is the most convenient way to monitor the efficiency of transfer Pre stained standards are protein markers that are covalently bound to a synthetic dye This enables visualization of the protein markers during electrophoresis and after the transfer Invitrogen offers three types of pre stained standards page 32 for ordering information The transfer efficiency is good if most of the standards have transferred to the membrane Note that high molecular weight standards do not always transfer completely and this is not indicative of incomplete transfer If none of the standards or only a few have transferred to the membrane you may have to optimize the transfer conditions Refer to Optimizing Blotting Parameters on the next page and the Troubleshooting section on page 23 Staining the gel The gel can be stained with Coomassie Blue staining or a staining method of choice after the transfer to determine the transfer efficiency If significant amount of proteins are still present on the gel after staining this indicates poor transfer You may need to optimize the transfer as described on
12. bic character and solvent resistant Physically stronger than nitrocellulose Compatible with commonly used protein stains and immunodetection methods Protein binding capacity 50 150 ug cm Microporous membrane modified with strongly basic charged groups Ideal for binding negatively charged biomolecules such as DNA and RNA Low background for enhanced resolution Membrane is formed around a non woven polyester fiber matrix which confers high tensile strength toughness and flexibility Protein sequencing Western transfer Amino acid analysis Solid phase assay systems 0 2 um Yes 0 45 um Southern northern and western transfers Solid phase immobilization Dry chemistry test strips Enzyme immobilization Gene probe assays Preparing for Transfer Introduction Materials Needed Z Bis Fs N c gE 2 NOT I Preparing Transfer Buffer You need to prepare the transfer buffer blotting pads and blotting membranes before performing the transfer You may prepare the transfer buffer and materials for transfer while electrophoresis of the gel is in progress e Pre cut blotting membranes and filter paper sandwiches e Methanol e Deionized water e Transfer buffer see below e Shallow tray for equilibration of membranes filter paper and blotting pads Wear gloves at all times during the entire blotting procedure to prevent contamination of gels and membranes and
13. easily inserted into the XCell SureLock Mini Cell in place of the gel buffer core assembly The module has rails to guide the unit into the mini cell The XCell IT Blot Module can be used to perform western Southern or northern transfer of two mini gels using only 200 mL of transfer buffer The XCell II Blot Module is a semi wet transfer unit An efficient transfer is obtained as the resistance is constant across the blotting electrodes producing uniform field strength Continued on next page Overview Continued Note The XCell II Blot Module can only be used with XCell SureLock Mini Cell or XCell IT Mini Cell Refer to page 32 for ordering information Blotting Membranes Nitrocellulose Invitrogen offers three types of blotting membrane and filter paper sandwiches Refer to page 32 for ordering information e Nitrocellulose for western or Southern blotting e PVDF polyvinylidene difluoride for western blotting e Nylon for Southern or northern blotting See the table below for more details on each membrane size Most widely used membrane for western blotting Good binding capacity Proteins bind to the membrane due to hydrophobic interactions Protein binding capacity 80 ug cm Western transfer Amino acid analysis Solid phase assay systems 0 2um No 0 45 um Continued on next page page Overview Continued size Higher binding capacity than nitrocellulose Strong hydropho
14. fer Expected Conditions Current Novex Tris Novex Tris Glycine Nitrocellulose 25 V constant Start Glycine Transfer Buffer with or PVDF for 1 2 hours 100 mA 20 methanol 1X Transfer Buffer should be pH 8 3 before addition of SDS or methanol Do not adjust the pH Novex Tricine NuPAGE NuPAGE Transfer Nitrocellulose 30 V constant Novex Bis Buffer with 10 or PVDF for 1 hour Tris methanol for transfer of one gel NuPAGE Antioxidant for reduced samples NuPAGE NuPAGE Transfer Nitrocellulose 30 V constant Novex Tris Buffer with 10 or PVDF for 1 hour Acetate methanol for transfer of one gel NuPAGE Antioxidant for reduced samples Novex TBE 45 mM Tris 30 V constant TBE Urea and 45 mM boric acid for 1 2 hours DNA Retardation 1 mM EDTA Novex IEF 0 7 acetic acid pH 3 0 Nitrocellulose 10 V constant Start 65 see page 27 or PVDF for 1 hour 85 mA Assemble the gel membrane sandwich in reverse order so that the membrane is on the cathode side of the gel page 27 Continued on next page 16 Using the XCell II Blot Module Continued Overnight Blotting Cleaning the Blot Module For overnight blotting perform transfer in the cold room with low power to prevent overheating Transfer at constant voltage of 10 15 V overnight Depending on the transfer efficiency adjust the transfer conditions accordingly Rinse the blot module with distilled water after
15. fer buffer 10 Using the XCell II Blot Module Introduction Materials Needed Removing the Gel The blotting protocol described below is suitable for majority of protein blotting applications using the XCell II Blot Module However some optimization may be necessary by the user to obtain the best results page 21 e Previously electrophoresed mini gels maximum gel size 9 cm x 9 cm e XCell SureLock Mini Cell Remove the gel from the cassette for transfer after completion of electrophoresis as described below If you are not ready to perform transfer immediately you may continue electrophoresis of your gel at a low voltage of 5 V The gel can be left in the unit for a few hours until you are ready to transfer the gel 1 After electrophoresis separate each of the 3 bonded sides of the gel cassette by inserting the gel knife into the gap between the cassette s 2 plates The notched well side of the cassette should face up 2 Push up and down 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 2 plates to avoid excessive pressure towards the gel 3 The gel may adhere to either side of plates upon opening the cassette Carefully remove and discard the plate without the gel The gel remains on the other plate Remove wells on the gel with the gel knife 5 Place a piece of p
16. fficiency 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 pl 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 It is more likely for native proteins to diffuse out of the membrane into the solution during the blocking or antibody incubation steps as the native proteins tend to be more soluble We recommend fixing the proteins to the membrane to prevent diffusion of the proteins The proteins can be fixed 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 methods the proteins will be sufficiently unfolded to expose hydrophobic sites and will bind more efficiently to the membrane Continued on next page 28 Special Applications Continued Semi Dry Blotting of NuPAGE N
17. he buffer is stable for 6 months at 25 C For transfer dilute the 25X Novex Tris Glycine Transfer Buffer as described on page 8 Continued on next page Buffer Recipes Continued 5X Novex 5X Novex TBE Running Buffer is available from Invitrogen TBE Running see next page Buffer 1 To prepare 5X Novex TBE Running Buffer dissolve the following reagents in 950 mL of deionized water Concentration 1X Tris Base 54 0 g 89 mM Boric Acid 27 5 g 89 mM EDTA free acid 2 9 g 2mM 2 Mix well and adjust the volume to 1 000 mL with deionized water The pH of the buffer is 8 3 3 Store the buffer at room temperature The buffer is stable for 6 months at 25 C 4 For transfer dilute the 5X Novex TBE Running Buffer as described on page 8 31 Accessory Products Additional Products Ordering information for electrophoresis products available separately from Invitrogen is provided below For detailed information visit the www invitrogen com or call Technical Support next page NuPAGE Novex 4 12 Bis Tris Gel Novex 10 Tris Glycine Gel XCell SureLock Mini Cell amp XCell IT Blot Module Sponge Pad for blotting 8 Gaskets 2 PowerEase 500 Power Supply Novex Sharp Pre Stained Protein Standard MagicMark XP Western Protein Standard SimplyBlue SafeStain Nitrocellulose Membrane 0 2 um pore size PVDF Membrane 0 2 um pore size Invitrolon PVDF Membrane 0 45 um pore size
18. hode core of the XCell II Blot Module to fit horizontally across the bottom of the unit There should be a gap of 1 cm at the top of the electrodes when the pads and assembly are in place see figure below lcm Blotting pads assembled horizontally 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 in only one way such that the sign is seen in the upper left hand corner of the blot module The inverted gold post on the right hand side of the blot module fits into the hole next to the upright gold post on the right side of the lower buffer chamber Continued on next page 13 Using the XCell II Blot Module Continued Transferring One Gel continued 14 Ts 10 11 Depending on the mini cell that you are using follow the appropriate instructions for positioning the wedge e For XCell SureLock Mini Cell place the gel tension wedge such that the vertical face of the wedge is against the blot module Push the lever forward to lock it into place e For XCell II Mini Cell place the front wedge without the screw hole such that the vertical face of the wedge is against the blot module Slide in the rear wedge and push it down firmly Note When properly placed the rear wedge will not be flush with the top of the lower buffer chamber There will be a gap between the rear wedge and
19. invitrogen XCell II Blot Module Catalog no El9051 Rev Date 14 December 2009 Manual part no IM9051 MAN0000740 Table of Contents Table of GofeNS aa ee 3 Product Contents and Specifications rrnnrrrrrnnnnnnnrrnnnnnnnr 4 OVE IGEN sadel EA AE EE E Re ise ential ded abet 5 Preparing for iiransl hsts nasa arena nts 8 Using the XCell II Blot Module cccccsesesesecseseeeeeeeeees 11 Testing the Efficiency of Blotting ceeeeeeeeeeeeeeeeeee 19 Optimizing Blotting ParameterS cccccceeeeeeeeeeeeeeeeeeeees 21 Troubleshooting cans dase beri datdeed donde bases ddaadlted dactetedahienid tut bis 23 Special Applications kassene 26 Buffer Recipe Seisean E aA n 30 Accessory Products rnnnnvrrrnnnnnnnnnrrnnnnnrrrnnnnnnnnnnrnnnnnrrnnnnnn 32 Technical Supportisrauruauemesusjeadjddde 33 Product Contents and Specifications Contents Specifications Intended Use The XCell II Blot Module includes anode core cathode core and sponge pad 8 pack The specifications of the XCell II Blot Module are Cell Dimensions 14 5 cm x 14cm x 11 cm Blot Module Capacity 200 mL XCell SureLock Lower Buffer Chamber Capacity 600 mL Blot Size 9 cm x 9 cm For research use only Not intended for any animal or human therapeutic or diagnostic use WEEE Waste Electrical and Electronic Equipment symbol indicates that this product should not be disposed of in unsorted municipal waste
20. les by rolling a glass pipette over the membrane surface Place the pre soaked filter paper on top of the transfer membrane Remove any trapped air bubbles Place 2 soaked blotting pads into the cathode core of the blot module The cathode core is the deeper of the 2 cores and the corresponding electrode plate is a darker shade of gray Carefully pick up the gel membrane assembly with your gloved hand and place on the pad in the same sequence such that the gel is closest to the cathode plate see figure below Hp Blotting Pad vee Gb r amp amp i Blotting Pad la a aa a aa 2 aa Filter Paper re 22020220 Transfer Membrane Filter Paper ua ae Blotting Pad Mee 0 e 0 0 eo ed Blotting Pad Cathode Core Continued on next page Using the XCell II Blot Module Continued Transferring 4 One Gel continued Add enough pre soaked blotting pads to rise 0 5 cm over the rim of the cathode core Place the anode core on top of the pads The gel membrane sandwich should be held securely between the two halves of the blot module ensuring complete contact of all components Note To ensure a snug fit use an additional pad since pads lose their resiliency after many uses Replace pads when they begin to lose resiliency and are discolored Position the gel membrane sandwich and blotting pads in the cat
21. lower chamber Fill the blot module with transfer buffer until the gel membrane sandwich is covered in transfer buffer Do not fill all the way to the top as this will generate extra conductivity and heat Fill the outer buffer chamber with 650 mL deionized water by pouring in the gap between the front of the blot module and 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 Note If you accidentally fill the outer buffer chamber with the transfer buffer it will not adversely affect the transfer The liquid in the outer buffer chamber serves as a coolant We recommend adding deionized water to the outer buffer chamber to avoid any exposure of the mini cell to methanol as the mini cell is susceptible to methanol Place the lid on top of the unit With the power turned off plug the red and black leads into the power supply Refer to Transfer Conditions on page 16 for transfer conditions Continued on next page Using the XCell II Blot Module Continued Transferring Two Gels Instructions are provided below for transferring two gels 1 2 Remove the gels after electrophoresis as described on page 11 Assemble the gel membrane sandwich as described on page 12 twice to make two gel membrane sandwiches Place two pre soaked pads on cathode core of the blot module Place the first gel
22. membrane sandwich on pads in the correct orientation so the gel is closest to the cathode plate see figure below d Blotting Pad eeeeee La as a a a aos a a a a GS G 8 6 Gb amp G Gb amp 1 A I a a 2 82 2 8 I Blotting Pad Filter Paper I EE I Transfer Membrane I a a ae a ee I Second Gel I I Filter Paper Se 0 6 6 6 6 6 6 6 0 a aa aa 4 Blotting Pad I I Filter Paper gt _ gt ___E_EE_ Transfer Membrane inna First Gel Filter Paper eeoeoeeeweeey i Pet ae ar Se i ge ae Ge Blotting Pad e e 0 o o o o o o o Blotting Pad l Cathode Core Add another pre soaked blotting pad on top of the first membrane assembly Position the second gel membrane sandwich on top of blotting pad in the correct orientation so that the gel is closest to the cathode side Proceed with Steps 4 11 as described in Transferring One Gel page 12 Continued on next page 15 Using the XCell II Blot Module Continued Transfer Choose the transfer conditions from the table below based Conditions on type of the gel that you are using Note The expected current listed in the table below is for transferring one gel If you are transferring two gels in the blot module the expected current will double For overnight blotting see next page Type of the Gel Transfer Buffer 1X Membrane Trans
23. ng 0 02 0 04 SDS for 10 minutes before assembling the sandwich 29 Buffer Recipes 20X NuPAGE Transfer Buffer 25X Novex Tris Glycine Transfer Buffer 30 20X NuPAGE Transfer Buffer is available from Invitrogen page 32 1 To prepare 20X NuPAGE Transfer Buffer dissolve the following reagents in 100 mL of deionized water Concentration 1X Bicine 10 2 g 25 mM Bis Tris free base 13 1g 25 mM EDTA 0 75 g 1mM Chlorobutanol 0 025 g 0 05 mM Mix well and adjust the volume to 125 mL with deionized water The pH of the buffer is 7 2 Store at room temperature The buffer is stable for 6 months at room temperature For transfer dilute the 20X NuPAGE Transfer Buffer as described on page 8 Chlorobutanol is used as a preservative in the transfer buffer and is not necessary for efficient transfer of proteins If you do not have chlorobutanol you may prepare the buffer without chlorobutanol but the buffer will not be stable for long periods Use the buffer within 2 weeks 25X Novex Tris Glycine Transfer Buffer is available from Invitrogen page 32 1 To prepare 25X Novex Tris Glycine Transfer Buffer dissolve the following reagents in 450 mL of deionized water Concentration 1X Tris Base 18 2 g 12 mM Glycine 90 0 g 96 mM Mix well and adjust the volume to 500 mL with deionized water The pH of the buffer is 8 3 Do not adjust with acid or base Store the buffer at room temperature T
24. nique on both membranes Monitor whether the primary membrane located next to the gel retains majority of the sample If the sample is detected on the membrane placed closer to the anode further away from the gel reduce the rate of transfer by lowering the field strength allowing more time for protein capture on the primary membrane Adjust the blotting protocol accordingly using the guidelines included in this section Troubleshooting Introduction No proteins transferred to the membrane Significant amount of protein is passing through the membrane indicated by the presence of proteins on the second membrane Significant amount of protein remains in the gel indicated by staining of the gel after transfer Review the information provided below to troubleshoot your experiments Gel membrane sandwich Assemble the sandwich in the assembled in a reverse direction such that proteins have migrated out into the solution Longer transfer time inappropriate SDS or methanol content or sample overloaded Shorter transfer time inappropriate gel type SDS or methanol content Higher molecular weight proteins usually do not transfer completely as compared to mid to low molecular weight proteins correct order using instructions provided on page 12 Re evaluate the percentage of the gel used Shorten the transfer time by 15 minute increments Remove any SDS which may have been added to the transfer b
25. of membranes as the background is high e 20 mL of SimplyBlue SafeStain page 32 with dry PVDF membranes and incubate for 1 2 minutes Wash the membrane three times with 20 mL of deionized water for 1 minute To avoid high background do not use SimplyBlue SafeStain on nitrocellulose and wet PVDF membranes e 0 5 Amido Black page 32 in 50 methanol and 10 acetic acid Remove excess stain with deionized water Destain with 45 methanol and 10 acetic acid for 30 minutes Rinse the membrane with deionized water and air dry e 0 1 Ponceau S page 32 in 7 trichloroacetic acid TCA for 5 minutes Rinse the membrane in deionized water to obtain transient staining or 10 acetic acid to obtain permanent staining and air dry e If you do not detect any proteins on the membrane after immunodetection or staining refer to the Troubleshooting section on page 23 Refer to the manufacturer s recommendations for optimizing immunodetection Testing the Efficiency of Blotting Introduction Once you have performed the transfer you may check the efficiency of transfer by using any one of the methods described below Testing the blotting efficiency helps you evaluate the transfer and optimize transfer parameters to obtain an efficient transfer We recommend testing the efficiency of blotting when you are performing the western transfer for the first time and if you have changed the buffer system or gel type Materials e Pre stained st
26. ovex Bis Tris Gels The NuPAGE Novex Bis Tris Gels do not transfer efficiently using a semi dry transfer cell as compared to blotting with XCell II Blot Module If you decide to use semi dry blotting for NuPAGE Novex Bis Tris Gels use the protocol provided below to ensure efficient transfer 1 Prepare 100 mL of 2X NuPAGE Transfer Buffer from 20X NuPAGE Transfer Buffer as follows NuPAGE Transfer Buffer 20X 10 0 mL NuPAGE Antioxidant reduced sample 0 1 mL Methanol 10 0 mL Deionized Water 79 9 mL Total Volume 100 mL If you are blotting large proteins see the Note below Soak the filter paper and transfer membrane in the transfer buffer e If you are using Novex pre cut membrane filter sandwiches use three filter papers 0 4 mm filter in thickness on each side of the gel or membrane e If you are not using the Novex pre cut membrane filter sandwiches use two thick filter papers Assemble the gel membrane filter paper sandwich on top of the anode plate as follows Filter Paper Filter Paper Filter Paper Membrane Gel Filter Paper Filter Paper Filter Paper Perform the transfer at 15 V constant for 15 minutes if you are using the Bio Rad Trans Blot Semi Dry Cell For any other semi dry transfer cell follow the manufacturer s recommendations Note For transfer of large proteins gt 100 kDa pre equilibrate the gel in 2X NuPAGE Transfer Buffer without methanol containi
27. re soaked filter paper as prepared on page 10 on top of the gel and lay just above the foot at the bottom of the gel leaving the foot of the gel uncovered Keep the filter paper saturated with transfer buffer and remove all trapped air bubbles by gently rolling over the surface using a glass pipette Continued on next page 11 Using the XCell II Blot Module Continued Removing the Gel continued Transferring One Gel 12 6 Turn the plate over so the gel and filter paper are facing downwards over a gloved hand or clean flat surface covered with a piece of Parafilm Remove gel from the plate using the following methods e Ifthe gel rests on the longer slotted plate use the gel knife to push the foot out of the slot in the plate and the gel will fall off easily e Ifthe gel rests on the shorter notched plate use the gel knife to carefully loosen the bottom of the gel and allow the gel to peel away from the plate 8 When the gel is on a flat surface cut the foot off the gel with the gel knife Proceed to Transferring One Gel below Note Once you have removed the gel from the unit and the cassette perform the transfer immediately Instructions are provided below for transferring 1 gel If you need to transfer 2 gels at a time refer to page 15 1 Wet the surface of the gel step 8 above with the transfer buffer and place pre soaked transfer membrane on the gel Remove air bubb
28. s as the porosity of the gel matrix is well matched with the different sizes of the proteins Some proteins can be equally well resolved on a Tricine gel or Tris Glycine gel In general a Tricine gel will resolve the same range of proteins as a higher percentage Tris Glycine gel and can be a better choice for some transfers The 1 0 mm thick gels are better for blotting Be sure to scale your sample load appropriately for the sensitivity of your antibody detection method A higher field strength volts cm may help larger proteins to transfer but may also cause smaller proteins to pass through the membrane without binding Our recommended condition for most proteins is 25 Volts for 90 minutes You may make minor adjustments 5 10V accordingly when necessary See Transfer Conditions page 16 Decreasing or eliminating alcohol may improve the transfer of some proteins especially large proteins For blotting Novex Tris Glycine Gels and 2 NuPAGE Novex Gels 20 methanol is added to the transfer buffer page 8 This is balanced by the residual SDS in the gel from the running buffer Keep this in mind when adjusting the methanol content in transfer buffer Continued on next page 21 Optimizing Blotting Parameters Continued Transfer Time SDS in Transfer Buffer Charge of Protein Hint 22 Increasing the transfer time to two hours will improve transfer of most proteins but may cause the smaller proteins
29. to pass through the membrane Transfer time usually has little influence on the detection of proteins that remain bound to the membrane Note that transfer times longer than 2 hours at the recommended power settings do not greatly improve transfer of proteins that have failed to transfer completely in 2 hours This may be due to the exhaustion of the buffer or partial fixation of the protein in the gel as a result of the removal of SDS or a conformational change in the proteins during the transfer interval Adding 0 01 to 0 02 SDS to the transfer buffer will facilitate the transfer of proteins especially large proteins but may reduce binding of proteins to the membrane especially nitrocellulose membranes In the blotting protocol on page 12 the gel is not incubated in the transfer buffer leaving residual SDS in the gel This is balanced by the 20 methanol added to the transfer buffer Keep this in mind when adjusting the SDS content in the transfer buffer For more basic proteins use a carbonate buffer The high pH of the buffer confers a higher negative charge on the more basic proteins and cause them to migrate faster Carbonate buffers improve binding detection for some systems Due to the high ionic strength of the carbonate buffer excessive heat may be generated during blotting We recommend using two membranes in tandem during initial blotting to closely monitor the protein transfer and then perform the same visualization tech
30. uffer If using nitrocellulose membrane switch to PVDF which has a higher binding capacity Add additional methanol to increase the binding capacity of the membrane Decrease the sample load Switch to a more appropriate lower percentage gel Increase the blotting time by 15 minute increments Add 0 01 0 02 SDS to the transfer buffer to facilitate migration of the protein out of the gel Decrease the amount of methanol in the transfer buffer Continued on next page 23 Troubleshooting Continued The pH of the Buffer not made up Remake the buffer after checking transfer buffer properly the reagents and water quality deviates from Do not adjust the pH with acid the required or base as this will increase the value by 0 2 pH conductivity of the buffer and units result in higher current during transfer Current is much Concentrated buffer used Dilute the buffer as described on higher than the page 8 expected start Used Tris HCI instead of Check the reagents used to make curent Tris Base the buffer and remake the buffer with correct reagents Currentis much Very dilute buffer used Remake the transfer buffer lower than the resulting in increased correctly expected start resistance and low current current The circuit is broken Check the blot module to ensure broken electrode that the electrodes are intact Leak in the blot module Be sure to assemble the blot indicated by a decrease in
31. use To clean any residual build up in the blot module apply 50 nitric acid in deionized water to areas inside the blot module until residual build up is removed Do not submerge the blot module or soak overnight in nitric acid Use gloves when preparing the nitric acid solution Once the build up is removed rinse the module at least three times in deionized water Continued on next page 17 Using the XCell II Blot Module Continued Post After the transfer you may proceed to immunodetection Transfer store the membrane for future use or stain the membrane Analysis e For immunodetection of proteins use the 18 WesternBreeze Chromogenic or Chemiluminescent Immunodetection Kits available from Invitrogen page 32 or any other immunodetection kit For storing nitrocellulose membranes air dry the membrane and store the membrane in an air tight plastic bag at room temperature or 4 C Avoid storing nitrocellulose at 20 C or 80 C as they will shatter For storing PVDF membranes air dry the membrane and store the membrane in a air tight plastic bag at room temperature 4 C or 80 C When you are ready to use the membrane re wet the membrane with methanol for a few seconds followed by thorough rinsing of the membrane with deionized water to remove methanol For staining the membranes after blotting you may use e 0 1 Coomassie Blue R 250 in 50 methanol Do not use Novex Colloidal Blue Staining Kit for staining
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