User Manual - Thermo Fisher Scientific
Contents
1. proteins to the binding capacity membrane Not enough methanol Increase the concentration of methanol in the transfer buffer Low molecular Use glutaraldehyde to crosslink the proteins weight proteins are to the membrane and use Tween 20 in the not binding well or wash steps are being washed away SDS is included in the Do not use SDS in the transfer buffer transfer buffer PVDF membrane Smeared or Membrane was dried Membrane should be completely gray and slightly translucent when added to the sandwich If it has dried out rewet in methanol and equilibrate in transfer buffer Alcohol was not used to prewet the membrane Membrane was not thoroughly wetted PVDF is hydrophobic and requires a short soak in methanol or ethanol prior to transfer Always pre wet the membrane according to the manufacturer s instructions White spots indicate dry areas of the membrane Too much current Use a low conductivity transfer buffer such as those recommended in this manual 11 Appendix Purchaser Notification Warranty Limited Use Label License No 5 Invitrogen Technology 12 Invitrogen warrants that this product will be free from defects in material and workmanship for a period of one 1 year from date of purchase If a defect is present Invitrogen will at its option repair replace or refund the purchase price of this product at no charge to you provided it is returned during the warranty2. blotting paper and also creates puddles that the current can pass through Continued on next page Troubleshooting continued Problem Cause Solution All membranes Insufficient binding of proteins to the membrane Air spaces are interfering with contact between the gel and the membrane Roll the membrane with a blotting roller or a clean test tube or pipet before placing the gel on the membrane then remove any air bubbles between the gel and membrane with a blotting roller or a wet gloved finger Transfer will not occur where the gel is not in contact with the membrane Electrophoretic conditions were incorrect or not ideal Running conditions sample preparation percentage acrylamide and many other variables can affect the migration and resolution of proteins Please review your electrophoresis conditions Under or over compression of gel Follow the compression guidelines in this manual Too little compression can allow proteins to migrate between the gel and membrane causing protein band smearing Too much compression can distort the gel Nitrocellulose membrane swirled transfer and missing bands Brown coloration of membrane or cracking of gel after transfer out before it was added to the transfer sandwich Insufficient Over transfer through Use 0 2 uM pore size nitrocellulose instead binding of the membrane of 0 45 uM or use PVDF with a higher
3. ml Deionized Water to 500 ml pH 8 3 1X Tris Acetate EDTA TAE Transfer Buffer A volume of 500 ml is sufficient for blotting 1 Midi Gel or 2 Mini Gels 10X Tris Acetate EDTA 0 4 Tris Acetate 0 01 disodium EDTA 50 ml Deionized Water to 500 ml pH 8 0 Continued on next page 5 Semi Dry Blotting Procedure continued Transfer Buffer for With its high buffering capacity and low ionic strength the following 1X NAQ Northern Blots Equilibrating the Gel Preparing the Blotting Membrane Gel Layout Northern Transfer Buffer is more efficient than TAE TBE or MOPS for transfer of RNA from agarose gels A volume of 500 ml is sufficient for blotting 1 Midi Gel or 2 Mini Gels 50X NAQ Northern Transfer Buffer 0 2 M MOPS 50 mM sodium acetate 5 mM EDTA pH 7 0 10 ml Deionized Water to 500 ml Equilibrating the gel in transfer buffer removes salts that may increase conductivity and heat during transfer Be careful to equilibrate for the recommended time as longer equilibration can result in protein diffusion 1 After electrophoresis remove the gel from the cassette 2 Place the gel in a shallow tray containing 100 ml for Midi Gels and E PAGE Gels or 50 ml for Mini Gels of the appropriate Transfer Buffer Equilibrate for 10 minutes on an orbital shaker E PAGE gels Remove the gel from the transfer buffer and gently rub a gloved finger over the well side of the gel to remove small gel pieces from the gel surface
4. period This warranty does not apply if the product has been damaged by accident abuse misuse or misapplication or from ordinary wear and tear For your protection items being returned must be insured against possible damage or loss This warranty shall be limited to the replacement of defective products It is expressly agreed that this warranty will be in lieu of all warranties of fitness and in lieu of the warranty of merchantability The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1
5. safety hazard Invitrogen is not responsible for any injury or damage caused by operating this blotter in a manner not specified in this manual All repairs and service should be performed by Invitrogen The Novex Semi Dry Blotter is classified as Class II for protection against electrical shock To ensure safe reliable operation always operate the Novex Semi Dry Blotter according to instructions provided in this manual Wear protective gloves and safety glasses when working in a laboratory environment Invitrogen is not responsible for injuries or damages caused by improper use The symbols used on the Novex Semi Dry Blotter are explained below Indicates an area on the blotter where a potential shock hazard may exist Indicates a warning Consult the user manual to avoid possible personal injury or instrument damage Product Components and Specifications List of The components included with the Novex Semi Dry Blotter are listed below Components Components Quantity Novex Semi Dry Blotter 1 Semi Dry Blotter Cable Red Anode 1 Semi Dry Blotter Cable Black Cathode 1 Semi Dry Blotter Knobs 3 Upon Receiving Examine the unit carefully for any damage incurred during transit Any damage the Instrument claims must be filed with the carrier The warranty does not cover in transit damage Novex Semi Dry Voltage Limit 50 VDC Blotter Dimensions 29x29 x 4 cm Specifications Transfer Area 21 x21 cm Shippin
6. 0 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 membranes after blotting you may use any total protein membrane staining methods such as Coomassie Blue R 250 Ponceau S Amido Black SYPRO Ruby Protein Blot Stain or SYPRO Rose Plus Protein Blot Stain page vii You may also use SimplyBlue SafeStain with dry PVDF membranes to avoid high background do not use SimplyBlue SafeStain on nitrocellulose and wet PVDF membranes If you do not detect any proteins on the membrane after immunodetection or staining refer to Troubleshooting on page 10 Refer to the immunodetection kit manufacturer s recommendations for optimizing immunodetection Troubleshooting Introduction 10 Review the information below to troubleshoot your experiments using the Novex Semi Dry Blotter Problem Cause Solution Transfer efficiency is poor Voltage is too low 1 mm thick polyacrylamide gels mini and Midi Gels should be transferred at 20 V E PAGE Gels at 25 V approximately 15 V cm field strength Power supply is inappropriate for semi dry transfer Transfer performed for too short a time Transfer sandwich was assembled in the wrong order Some power supplies will shut off or blow a fuse when run at the conditions required for semi dry transfer Semi dry tran
7. Re submerge the gel in transfer buffer to rinse away any gel pieces adhering to the gel as they can cause air bubbles and field distortion during transfer Nitrocellulose or Nylon 1 Use a pre cut membrane or cut a membrane to the appropriate size for your gel 2 Soak the membrane in the appropriate Transfer Buffer for a few minutes in a shallow tray PVDF 1 Use a pre cut Invitrolon Filter Paper Sandwich or cut a PVDF membrane to the appropriate size for your gel 2 Pre wet the membrane for 30 seconds in methanol ethanol or isopropanol Briefly rinse the membrane in deionized water 3 Soak the membrane in the appropriate Transfer Buffer for a few minutes in a shallow tray We recommend using the following gel layouts in the blotter to ensure even pressure of the plates on the gel stack 5 o 00 00 oo joe Single gel Mini or Midi Two gels Mini or Midi Three gels Mini Four gels Mini Continued on next page Semi Dry Blotting Procedure continued Compression of the Gel Stack Semi Dry Blotting Protocol If you are blotting only one Mini Gel the weight of the cathode plate lid alone will apply enough pressure to maintain good gel membrane contact If you are blotting Midi Gels or two or more Mini Gels it is necessary to slightly tighten the knobs on the Novex Semi Dry Blotter to ensure even pressure of the plates on the gel stack s Tighten the knobs as described in the following proto
8. Western transfer solvent resistant Amino acid Physically stronger than analysis nitrocellulose Solid phase assay Compatible with commonly used systems protein stains and immunodetection methods Protein binding capacity 50 150 pg cm Microporous membrane modified Southern and 0 45 um with strongly basic charged northern groups transfers Ideal for binding negatively charged Solid phase biomolecules such as DNA and immobilization RNA Dry chemistry Low background for enhanced test strips resolution Enzyme Membrane is formed around a non immobilization woven polyester fiber matrix Gene probe which confers high tensile assays strength toughness and flexibility Methods Semi Dry Blotting Procedure Introduction Materials Needed N N ND I Y NE o iP Northern and Southern Blotting This section provides guidelines and a protocol for the western blotting of proteins It also includes buffer formulations and information for northern and Southern blotting Methanol Transfer Buffer see buffer recipes on the following pages for details NuPAGE Antioxidant for reduced samples Blotting membranes o Nitrocellulose or PVDF membrane for western transfers TM Nitrocellulose Filter Paper Sandwiches and Invitrolon Filter Paper Sandwiches are available separately from Invitrogen o Nylon membrane for northern or Southern transfers available separately from Invitrogen 4 pieces of 2 5 mm thick Blott
9. buffer soaked filter paper that function as the ion reservoir During electrophoretic transfer negatively charged molecules migrate out of the gel and move towards the positive electrode where they are deposited on the membrane The plate electrodes separated only by the gel and filter paper stack provide high field strength V cm across the gel allowing for very efficient rapid transfers The Novex Semi Dry Blotter is a semi dry blotting system that allows you to quickly and easily perform simultaneous western blotting of up to 2 Midi Gels 4 Mini Gels or 2 E PAGE gels It can also be used to perform northern RNA and Southern DNA blotting The Novex Semi Dry Blotter is designed for fast set up and ease of use The platinum coated titanium anode plate and stainless steel cathode plate provide fast and efficient electroblotting using less transfer buffer than wet tank blotting methods Novex Semi Dry Blotter Lid Cathode Plate Anode Plate Black Cathode Cable Red Anode Cable Continued on next page 1 Overview continued Features Novex Semi Dry Blotter Cables Power Supply Requirements Blotting Membranes The Novex Semi Dry Blotter has the following features e Simple easy to use design e Allows simultaneous transfer of 1 2 Midi Gels 1 4 Mini Gels or 1 2 E PAGE gels e Requires less transfer buffer than wet blotting methods e Connector design prevents incorrect cable hook
10. col It is important that the pressure on the gel stack s be even without being too firm Tighten the knobs roughly evenly with regard to torque not necessarily with regard to rotational distance Too little compression can allow proteins to migrate between the gel and membrane causing protein band smearing Too much compression can distort the gel You can apply a small piece of tape to one of the bumps on each knob to make it easier to determine the rotational distance Follow the instructions below to blot 1 2 Midi Gels or 1 4 Mini Gels using the Novex Semi Dry Blotter 1 Ina shallow tray briefly soak 2 stacked pieces of 2 5 mm thick Blotting Filter Paper in the appropriate Transfer Buffer Several pieces of thinner blotting paper can be used to produce a stack of equivalent thickness 2 Remove any air bubbles trapped between the filter paper sheets by rolling the stack with a blotting roller while it is still submerged in buffer Note Removing air bubbles is essential as they can block the transfer of biomolecules 3 Place the stack of pre soaked Blotting Filter Paper on the anode plate of the Novex Semi Dry Blotter Remove any air bubbles between the paper and plate by rolling the stack with the blotting roller 4 Place the pre soaked blotting membrane on top of the Blotting Filter Paper stack and remove any air bubbles with the blotting roller 5 Carefully remove the gel from the transfer buffer and place on top of
11. er volume of liquid NuPAGE Novex Bis Tris or Tris Acetate Gels and E PAGE 48 Gels Prepare the following 2X NuPAGE Transfer Buffer This transfer buffer maintains the neutral pH environment established during gel electrophoresis protects against modification of the amino acid side chains and is compatible with N terminal protein sequencing using Edman degradation A volume of 500 ml is sufficient for blotting 1 Midi Gel or 2 Mini Gels Reduced Non reduced NuPAGE Transfer Buffer 20X 50 ml 50 ml NuPAGE Antioxidant 0 5 ml Methanol 50 ml 50 ml Deionized Water to 500 ml to 500 ml E PAGE 96 Gels Prepare the following 2X NuPAGE Transfer Buffer without methanol A volume of 500 ml is sufficient for blotting 1 E PAGE 96 gel Reduced Non reduced NuPAGE Transfer Buffer 20X 50 ml 50 ml NuPAGE Antioxidant 0 5 ml Deionized Water to 500 ml to 500 ml Novex Tris Glycine or Tricine Gels Prepare the following 2X Tris Glycine Transfer Buffer A volume of 500 ml is sufficient for blotting 1 Midi Gel or 2 Mini Gels Tris Glycine Transfer Buffer 25X 40 ml Methanol 50 ml Deionized Water to 500 ml Prepare the appropriate transfer buffer for DNA agarose and polyacrylamide gels from the recipes below 1X Tris Borate EDTA TBE Transfer Buffer A volume of 500 ml is sufficient for blotting 1 Midi Gel or 2 Mini Gels Note that a 0 5X concentration may also be used Novex TBE Running Buffer 5X 100
12. g Filter Paper 2 5 mm thick 7 5 x 8 4 cm 50 LC2010 Nitrocellulose membrane 0 20 um pore size 7 3 x 8 3 cm 20 LC2000 0 45 um pore size 7 3 x 8 3 cm 20 LC2001 Invitrolon PVDF Filter Paper Sandwich 0 45 um pore size 8 3 x 7 3 cm 20 LC2005 0 20 um pore size 8 3 x 7 3 cm 20 LC2002 Nylon membrane filter paper sandwich 0 45 um pore size 8 3 x 7 3 cm 20 LC2003 Buffers and Reagents NuPAGE Antioxidant 15 ml NP0005 NuPAGE Tris Glycine Transfer Buffer 20X 125 ml NP0006 1L NP0006 1 NuPAGE Transfer Buffer 25X 500 ml LC3675 Novex TBE Running Buffer 5X 1L LC6675 Staining SimplyBlue SafeStain 1L LC6060 SYPRO Ruby Protein Blot Stain Kit 10 40 S 11791 blots SYPRO Rose Plus Protein Blot Stain Kit 10 40 S 12011 blots Continued on next page vi Additional Products continued Additional Products continued Immunodetection Quantity Catalog no WesternBreeze Chromogenic Western Blot Detection Kit 1 Kit WB7103 aMouse 1 Kit WB7105 aRabbit WesternBreeze Chemiluminescent Western Blot Detection 1 Kit WB7104 Kit aMouse 1 Kit WB7106 aRabbit Other Hardware Blotting roller 8 6 cm wide 1 LC2100 Incubation tray 10 x 14 cm 8 LC2102 Gel Knife 1 E19019 ZOOM Dual Power Supply 100 120 VAC 50 60 Hz 1 ZP10001 ZOOM Dual Power Supply 220 240 VAC 50 60 Hz 1 ZP10002 PowerEase 500 Power Supply 100 120 VAC 50 60 Hz 1 El8600 PowerEase 500 P
13. g Weight 8 pounds Lid Material Acrylic Base Material Acrylic Anode Conductive Plate Platinum coated titanium Cathode Conductive Plate Polished stainless steel Cables Insulated copper conductors Operating Temperature 4 40 C Do not use ethanol or other organic solvents to clean the Novex Semi Dry Blotter Organic solvents can cause acrylic to crack Rinse the blotter with deionized water after each use to clean and let air dry If you must wipe the surface take particular care not to scratch or otherwise damage the platinum coated titanium plate Important vi Additional Products Additional Products Additional products for western blotting are available separately from Invitrogen Ordering information is provided below For more information visit our Web site at www invitrogen com or call Technical Service page 12 Blotter Spare Parts Quantity Catalog no Novex Semi Dry Blotter Cable Red 1 SD0004 Novex Semi Dry Blotter Cable Black 1 SD0005 Novex Semi Dry Blotter Knob 1 SD0006 Novex Semi Dry Blotter Screw 1 SD0007 Filter Papers and Membranes Midi Gels and E PAGE gels Blotting Filter Paper 2 5 mm thick 8 6 x 13 5 cm 50 LC2008 Nitrocellulose membranes 0 20 um pore size 8 5 x 13 5 cm 20 LC2009 0 45 um pore size 8 5 x 13 5 cm 20 LC2006 Invitrolon PVDF Filter Paper Sandwich 0 45 um pore size 8 6 x 13 5 cm 20 LC2007 Mini Gels Blottin
14. ing Filter Paper per gel available separately from Invitrogen or equivalent pieces of thinner blotting filter paper E PAGE Gels only E PAGE Blotting Pads included with gels Blotting roller available separately from Invitrogen Deionized water Shallow trays for equilibrating membranes gels filter paper and blotting pads Incubation Trays are available separately from Invitrogen Gel Gel knife Wear gloves at all times during the entire blotting procedure to prevent contamination of gels and membranes to avoid exposure to irritants commonly used in electrophoresis and blotting procedures and to avoid staining the anode plate with oils in your skin The platinum plated titanium anode plate is easily scratched and should be handled with care Do not adjust the pH of the Transfer Buffer as this may alter the conductivity of the solution Northern and Southern blotting may be performed using the same blotter components and configuration lid cables knobs as western blotting No special blotter components are required Continued on next page Semi Dry Blotting Procedure continued Transfer Buffer for Western Blots Transfer Buffer for Southern Blots Prepare the appropriate transfer buffer for your gel type from the recipes below For semi dry western blotting the transfer buffer must be at twice the concentration used in wet blotting i e 2X to ensure that there are enough buffering ions present in the small
15. invitrogen Novex Semi Dry Blotter For semi dry electroblotting of proteins from polyacrylamide and agarose gels Catalog no SD1000 Version A 10 July 2006 25 0949 ii Table of Contents A O v Product Components and Specifications occcononcncnnonononenenennnnnnnnrornnnnnnnnnanananananonnnnnranananonnr nana nana rarananannno vi Additional Product tata vii INIFOQUEHON cian EE EE EE 1 Overview RN 1 AUAA A O T 4 Semi Dry Blotting Procedure cursa ndo 4 ROSt LAWS LOLS A AR tea ewes 9 Trouble Oi a RR EAE ltda des 10 APpeNdIX A 12 Purchas r Noticas iD 12 Technical SUppOlbivvmivininia alado ea 13 iii iv Safety Information Safety Important Informational Symbols A A Only use the Novex Semi Dry Blotter with a ground isolated external DC power supply This power supply should be equipped with No Load or Open Circuit protection Operating limits for the Novex Semi Dry Blotter are Maximum Voltage Limit 50 Volts DC Operating Temperature 4 40 C The Novex Semi Dry Blotter is designed to be operated with the lid in place If the lid is removed the electrical connection will be broken Do not attempt to use the blotter without the lid in place The Novex Semi Dry Blotter is safe to use when operated in accordance with the instructions in this manual Do not use or modify the blotter in a manner not specified in this manual Alteration of the blotter will e Void the warranty e Create a potential
16. ir bubbles Briefly soak the remaining 2 stacked pieces of 2 5 mm thick Blotting Filter Paper in the appropriate Transfer Buffer Remove any air bubbles with the blotting roller while the stack is still submerged in buffer Place the stack of pre soaked Blotting Filter Paper on the gel Ensure that the filter paper sheets are aligned properly and flush with the gel membrane sandwich Gently remove any air bubbles with the blotting roller Repeat Steps 1 8 for each gel you are blotting When finished make sure there are no puddles of buffer on the anode plate Place the cathode plate lid on the stack taking care to properly align the lid holes with the screws and the anode plate electrical connector Be careful not to disturb the blot sandwich If you are blotting only one Mini Gel the weight of the cathode plate lid alone will apply enough pressure to maintain good gel membrane contact For two or more Mini Gels or if you are blotting Midi Gels or E PAGE gels thread the knobs onto the blotter screws and spin them down until they just make contact with the lid Then tighten the knobs another to 1 turn depending on the number of gels in the blotter the greater the number of gels the more tightening required Note Tighten the knobs roughly evenly with regard to torque not necessarily with regard to the number of turns Do not overtighten Connect the power cables from the blotter to the power supply making sure that the red
17. ower Supply 220 240 VAC 50 60 Hz 1 EI8700 Related Instruments iBlot Dry Blotting System 1 IB1001 IB1001UK IB1001EU XCell IT Blot Module 1 EI9051 Pre cast Gels and Pre made Buffers Other Blotting Systems from Invitrogen viii A large variety of pre cast gels including NuPAGE Novex Tris Glycine Mini and Midi Gels and E PAGE Gels is available from Invitrogen For details contact Technical Support page 13 or visit www invitrogen com Invitrogen also has the following blotting systems TM The iBlot Dry Blotting System allows for fast dry western blotting of proteins from various types of gels without the need to prepare buffers The proteins transferred using the iBlot Dry Blotting System exhibit higher immunodetection sensitivity compared with proteins transferred using conventional semi dry or semi wet blotting methods The XCell II Blot Module is a semi wet system for blotting up to two Mini Gels and is designed to be inserted into the XCell SureLock Mini Cell in place of the gel buffer core assembly Overview Introduction Disassembled Assembled Introduction Western electroblotting is the electrophoretic transfer of separated proteins in a gel to the surface of a thin support membrane matrix where they are bound and immobilized Semi dry blotting is performed with plate electrodes in a horizontal configuration sandwiching a gel and membrane between sheets of
18. sfer requires low voltage 20 V and high current Check with the manufacturer of the power supply to determine whether it is appropriate for semi dry transfer Increase the amount of time for transfer Typical transfer times range from 30 to 60 minutes The Novex Semi dry Blotter is configured with the cathode on the top and anode on the bottom This results in a downward transfer of proteins from the gel onto the membrane Follow the instructions carefully when assembling the transfer sandwich The pH of the transfer buffer is too close to the isoelectric point of the protein The transfer buffers should be at the optimal pH if prepared as described in this manual Do not adjust the pH with acid or base as this will increase the conductivity of the buffer and result in higher current during transfer Too much methanol in the transfer buffer Reducing the amount of methanol can help elute proteins from the gel but can reduce binding to nitrocellulose membranes High percentage gels restrict transfer Higher percentage acrylamide or crosslinkers can restrict elution of proteins Use the lowest percentage acrylamide possible to separate your proteins Puddles of buffer were present on the plates allowing the current to bypass the stack Always clean up the lower plate before closing the lid of the transfer apparatus Do not squeeze the stack excessively as this removes transfer buffer from the
19. tations handbooks etc e Complete technical service 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 fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive f Paisley PA4 9RF UK Tel 1 760 603 7200 Minato ku Tokyo 108 0022 me Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 A Fax 81 3 5730 6519 E mail tech_support invitrogen com E mail E mail eurotech invitrogen com jpinfo invitrogen com 2006 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 13 Notes 8 invitrogen Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech service invitrogen com For country specific contact information visit our web site at www invitrogen com
20. the blotting membrane E PAGE gels Place the flat side of the gel on the blotting membrane such that the well side of the gel is facing up Gently remove any air bubbles with the blotting roller or a wet gloved finger Note Be careful not to disturb the gel after it has been placed on the membrane Moving the gel can result in protein smearing on the membrane Optional To physically support the gel making it easier to pick up and move place a piece of thin filter paper included with the membrane filter paper sandwiches from Invitrogen on top of the gel in the transfer buffer Do not fully submerge the paper in buffer a small amount of buffer on top of the gel will allow the paper to lightly adhere to the gel surface Then carefully slide a gel knife under one corner of the gel and lift the gel filter paper out of the tray After the gel has been placed on the blotting membrane gently peel away the thin filter paper Protocol continued on next page Continued on next page Semi Dry Blotting Procedure continued Semi Dry Blotting Protocol continued Protocol continued from previous page 6 10 11 12 13 14 E PAGE Gels only Fill the wells of the gel with Transfer Buffer after it has been placed on the stack Soak the E PAGE Blotting Pad in Transfer Buffer forcing air out of the pad by pressing it with a gloved finger and TM then place E PAGE Blotting Pad on the gel and gently roll out a
21. to red and black to black connections are correct Mini or Midi Gels Transfer at 20 V for 30 60 minutes E PAGE Gels Transfer at 25 V for 30 60 minutes The transfer time may vary depending on the properties of the proteins and should be determined empirically Proceed to Post Transfer next page Post Transfer Post Transfer Disassembly and Cleaning Post Transfer Analysis To disassemble the blotter and gel stack 1 2 Remove the knobs and lift off the lid of the Novex Semi Dry Blotter Carefully separate the membrane from the blotting stack and proceed with post transfer analysis see below The gel may be retained for post transfer staining Rinse the Novex Semi Dry Blotter with deionized water and air dry after each use For repairs and service contact Technical Service page 12 After transfer you may proceed to immunodetection store the membrane for future use or stain the membrane For immunodetection of proteins use the WesternBreeze Chromogenic or Chemiluminescent Immunodetection Kits available from Invitrogen page vii or any other immunodetection kit For storing nitrocellulose membranes air dry the membrane and store the membrane in an air tight plastic bag or dish at room temperature or 4 C Avoid storing nitrocellulose below 20 C as it may turn brittle For storing PVDF membranes air dry the membrane and store in an air tight plastic bag or dish at room temperature 4 C or 8
22. ups The Novex Semi Dry Blotter cables will fit onto the blotter in only one orientation to prevent incorrect connection to the electrodes The black cathode cable has a white acrylic safety ring attached to the end to prevent connection to the recessed red anode connector The Novex Semi Dry Blotter is used with an external DC power supply that must e Be isolated from the ground so that the DC output is floating e Be equipped with No Load or Open Circuit protection e Be able to operate at 20 V The ZOOM Dual Power Supply and PowerEase 500 Power Supply are compatible for use with this blotter See page vii for ordering information Invitrogen offers three types of blotting membrane and filter paper sandwiches Refer to page vii 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 following table for more details on each membrane Protein Pinding capacity 80 pg cm Nitrocellulose Most widely used membrane for Western transfer 0 2 um western blotting Amino acid 0 45 um Good binding capacity analysis Proteins bind to the membrane due to Solid phase assay hydrophobic interactions systems Continued on next page page Overview continued Blotting Membranes continued Higher binding capacity than Protein 0 2 um Yes nitrocellulose sequencing 0 45 pm Strong hydrophobic character and
23. use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Invitrogen Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Invitrogen Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Invitrogen is willing to accept return of the product with a full refund For information on purchasing a license to this product for purposes other than research contact Licensing Department Invitrogen Corporation 1600 Faraday Avenue Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 Email outlicensing invitrogen com Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations ci
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