Mini Trans-Blot® Electrophoretic Transfer Cell Instruction Manual
Contents
1. A rtisan Artisan Technology Group is your source for quality Fra op new and certified used pre owned equipment FAST SHIPPING AND SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT DELIVERY Experienced engineers and technicians on staff Sell your excess underutilized and idle used equipment TENS OF THOUSANDS OF at our full service in house repair center We also offer credit for buy backs and trade ins IN STOCK ITEMS www artisantg com WeBuyEquipment 7 EQUIPMENT DEMOS HUNDREDS OF InstraV ea REMOTE INSPECTION LOOKING FOR MORE INFORMATION MANUFACTURERS Remotely inspect equipment before purchasing with Visit us on the web at www artisantg com 7 for more our interactive website at www instraview com information on price quotations drivers technical LEASING MONTHLY specifications manuals and documentation RENTALS ITAR CERTIFIED EE Contact us 888 88 SOURCE sales artisantg com www artisantg com Mini Trans Blot Electrophoretic Transfer Cell Instruction Manual Catalog Numbers 170 3930 170 3935 For Technical Service Call Your Local Bio Rad Office or in the U S Call 1 800 4BIORAD 1 800 424 6723 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Note Assembly and Disassembly To insure best performance from the Mini Trans Blot electrophoretic transfer cell become fully acquainted with these operating instructions before using t2. ting does not occur immediately by immersion of the sheet in transfer buffer heat distilled water until just under the boiling point and soak the membrane until com pletely wet Equilibrate in transfer buffer until ready for use Poor binding to the membrane PVDF 1 The membrane may not be completely wet e Because of the hydrophobic nature of PVDF the membrane must be prewet in alco hol prior to equilibration in aqueous transfer buffer Follow the directions in the product insert 2 The membrane may have been allowed to dry during handling e A completely wet membrane has a gray translucent appearance White spots will form on the surface of the membrane indicating that it has been allowed to dry Since proteins will not bind to the dry spots rewet the membrane with methanol and re equilibrate in transfer buffer 6 2 Immune Specific Detection Overall high background 1 Blocking conditions are inappropriate e Match the blocker to the membrane For example nylon and PVDF membranes require more extensive blocking usually with non fat dry milk e Increase the concentration or blocking time as necessary e The blocker must be a pure protein The blocker may be contaminated with materi al that binds probes non specifically 2 Insufficient wash protocols are used e Increase the number duration or stringency of the washes Include progressively stronger detergents in the washes e g SDS is stronger than NP 40 which is
3. Add a second sheet of membrane to bind excess protein The transfer buffer is contaminated e Make fresh solutions Poor binding to the membrane Nitrocellulose 1 Nitrocellulose requires 20 methanol in the transfer buffer for optimal protein binding e Make sure the buffer contains the proper amount of methanol Proteins may be transferring through the nitrocellulose e Use PVDF or nylon higher binding capacities or 0 2 um nitrocellulose smaller pore size Decrease the voltage or move the electrodes to the standard position if using the high intensity option Mixed ester celluloses bind proteins poorly e Use pure nitrocellulose Proteins lt 15 000 daltons may show diminished binding to 0 45 um nitrocellulose or may be washed from the membrane during assays e To increase stability of binding proteins can be crosslinked to nitrocellulose with glutaraldehyde e Use PVDF or nylon membrane which have higher binding capacities e Use Tween 20 detergent in the wash and antibody incubation steps Reduce or elim inate the more stringent washing conditions SDS in the transfer buffer will reduce binding efficiency of proteins e Reduce or eliminate the SDS from the transfer buffer 17 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 6 The membrane may not be completely wet e White spots on the membrane indicate dry areas where protein will not bind If wet
4. crosslinker A 5 C with bis as the crosslinker will produce the smallest pore size gel Decreasing from this concentration will increase the pore size and increase transfer efficiency Poor transfer nucleic acid 1 Gel percentage is too high e Reduce the T or C in the acrylamide gel or reduce agarose in an agarose gel e Prior to transfer cleave DNA in dilute 0 25 M HCl or RNA in dilute NaOH 2 Transfer time is too short or power conditions are too low e Increase the transfer time or try high intensity transfer 3 DNA or RNA cannot be transferred electrophoretically to nitrocellulose since high salt concentrations are required for efficient binding e Use Zeta Probe membrane instead of nitrocellulose Swirls or missing bands diffuse transfers 1 Poor contact between the membrane and the gel Air bubbles or excess buffer remain between the blot and gel e Use atest tube or pipet as a rolling pin and roll over the membrane carefully in both directions until air bubbles and excess buffer are removed from between gel and membrane and complete contact is established e Use thicker filter paper in the gel membrane sandwich e Replace the fiber pads Pads will compress with time and will not hold the mem brane to the gel 2 Power conditions are too high e Always check the current at the beginning of the run The current may be too high for a particular voltage setting If the buffer is prepared improperly the co
5. 114 569 1981 Burnette W N Anal Biochem 112 195 1981 Legocki R P and Verma D P S Anal Biochem 111 385 1981 Lin W and Kasamatsu H Anal Biochem 128 302 1983 Anderson N L Nance S L Pearson T W and Anderson N G Electrophoresis 3 135 1982 McLellan T and Pamshaw J A M Biochem Genetics 19 647 1981 Gibson W Anal Biochem 118 1 1981 23 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 23 Howe J G and Hershey J W B J Biol Chem 2566 12836 1981 24 Erickson P G Minier L N and Lasher P S J Immun Meth 51 241 1982 25 Tsang V C W Peralta J M and Simons A R Meth Enzymol 92 377 1983 26 Gershoni J M and Palade G E Anal Biochem 124 396 1982 27 Gershoni J M and Palade G E Anal Biochem 131 1 1983 28 Symington J Two Dimensional Gel Electrophoresis of Proteins Methods and Applications Celis J E and Bravo R eds Academic Press N Y 1983 29 Andrews A T Electrophoresis Theory techniques and biochemical and clinical application 2nd ed Clarendon Press Oxford 1986 30 Beisiegel V Electrophoresis 7 1 1986 31 Bio Rad Laboratories unpublished 32 Gershoni J M in Advances in Electrophoresis Vol 1 Chrambach A Dunn M J and Radola B J eds VCH Weinheim in press 33 Gersh
6. Bio Ice cooling unit keep frozen at 20 C Buffer tank Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 2 2 Preparation for Blotting Fill the Bio Ice cooling unit with water and store it in your laboratory freezer at 20 C until ready to use After use return the cooling unit to the freezer for storage 1 Prepare the transfer buffer See Section 3 3 for buffer formulation Using buffer chilled to 4 C will improve heat dissipation 2 Cut the membrane and the filter paper to the dimensions of the gel Always wear gloves when handling membranes to prevent contamination Equilibrate the gel and soak the membrane filter paper and fiber pads in transfer buffer 15 min 1 hour depending on gel thickness 3 Prepare the gel sandwich Place the cassette with the gray side down on a clean surface Place one pre wetted fiber pad on the gray side of the cassette Place a sheet of filter paper on the fiber pad Place the equilibrated gel on the filter paper Place the pre wetted membrane on the gel Complete the sandwich by placing a piece of filter paper on the membrane Add the last fiber pad Removing any air bubbles which may have formed is very important for good results Use a glass tube to gently roll air bubbles out Fiber pad Filter paper Membrane Gel Filter paper Fiber pad Artisan Technology Group Quality Instru
7. Transfer apparatus is assembled incorrectly and the proteins are moving in the wrong direction e The gel membrane sandwich may be assembled in the wrong order or the cassette is inserted in the tank facing the opposite orientation Check the polarity of the con nections to the power supply Charge to mass ratio is incorrect e Try amore basic or acidic transfer buffer to increase protein mobility Proteins near their isoelectric point at the pH of the buffer will transfer poorly It has been suggested that buffer pH should be 2 pH units higher or lower than the pI of the protein of inter est for optimal transfer efficiency 15 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 5 Protein is precipitating in the gel e Try using SDS in the transfer buffer SDS can increase transfer efficiency but can also reduce binding efficiency to nitrocellulose and affect reactivity of some proteins with antibodies 6 Power supply circuit is inoperative or an inappropriate power supply was used e Check the fuse Be sure the voltage and current output of the power supply match the needs of the blotting instrument 7 Methanol in the transfer buffer is restricting elution e Reduction of methanol results in increased transfer efficiency of proteins from the gel but it also diminishes binding to nitrocellulose and PVDF 8 Gel percentage too high e Reduce T total monomer or C
8. lid assembly providing a safety interlock to the user Current to the the cell is broken when the lid is removed Do not attempt to circumvent this safety interlock and always turn the power supply off before removing the lid or when working with the cell in any way Current to the cell provided from the external power supply enters the unit through the Important This Bio Rad instrument is designed and certified to meet IEC 1010 1 safe ty standards Certified products are safe to use when operated in accordance with the instruction manual This instrument should not be modified or altered in any way Alteration of this instrument will e Void the manufacturer s warranty e Void the IEC1010 1 safety certification e Create a potential safety hazard Bio Rad is not responsible for any injury or damage caused by the use of this instrument for purposes other than for which it is intended or by modifications of the instrument not per formed by Bio Rad or an authorized agent TEC 1010 1 is an internationally accepted electrical safety standard for laboratory instruments Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 2 Mini Trans Blot Cell Assembly and Preparation for Transfer 2 1 Mini Trans Blot Cell Description and Assembly of Parts Lid Fiber pad Filter paper Membrane Gel Filter paper Fiber pad Gel holder cassette Electrode module
9. of the experiment The physical properties and performance char acteristics of a membrane should be evaluated when selecting the appropriate transfer conditions Binding Capacity Membrane Pore Size ug em Notes Nitrocellulose 0 45 um 80 100 General purpose protein blotting membrane 0 2 um Supported 0 45 um 80 100 Pure nitrocellulose cast on an inert synthetic support Nitrocellulose 0 2 um increased strength for easier handling and for reprobing PVDF 0 2 um 170 200 High mechanical strength and chemical stability used for protein sequencing and western blotting enhanced binding in the presence of SDS Must be wet in alcohol before equi libration in buffer Nylon 0 2 um 170 Recommended for nucleic acids Note Nucleic acids cannot be transferred to nitrocellulose by electrophoretic blotting Use Zeta Probe membrane Section 6 Troubleshooting Guide 6 1 Electrophoretic Transfer Poor electrophoretic transfer as detected by staining the gel proteins 1 Transfer time is too short e Increase the transfer time Power is too low e Always check the current at the beginning of the run The current may be too low for a particular voltage setting If the buffer is prepared improperly the conductivi ty may be too low and not enough power will be delivered to the cell See the power guidelines for specific applications in Section 3 e Remake the buffer or increase the voltage e Try the high intensity blotting option
10. stronger than Tween 20 Also include Tween 20 in the antibody dilution buffers to reduce non specific binding 3 The blot is left in the substrate too long e Remove the blot from the substrate solution when the signal to noise level is accept able Do not overdevelop Stop the reaction immediately by immersing the blot in dd H O 2 4 Contamination occurred during a previous step e g electrophoresis or transfer e Discard and remake the gel and transfer solutions e Replace or thoroughly clean contaminated fiber pads Excessive amounts of protein were loaded on the gel or too much SDS was used in the transfer buffer Proteins can pass through the membrane without binding and recirculate through a tank blotting system Reduce the amount of protein on the gel or SDS in the transfer buffer Add a second sheet of membrane to bind excess protein 18 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 5 Primary or secondary antibody is too concentrated e Increase the dilution of the antibodies Perform a dot blot experiment to optimize the working concentrations 6 Incubation trays are contaminated e Clean the trays or use disposable trays Nonspecific reactions between bound proteins and probes 1 Primary or secondary antibody is contaminated with nonspecific or species cross reactive IgG e Use purified IgG first antibody fractions and affinity purified blotting gr
11. the pH electrode functions properly for Tris buffers and the pH is below 8 0 remake the buffer 25 mM Tris 192 mM glycine 20 v v methanol pH 8 3 Mix 3 03 g Tris 14 4 g glycine and 200 ml of methanol add distilled deionized water dd H O to 1 liter 25 mM Tris 192 mM glycine pH 8 3 Mix 3 03 g Tris and 14 4 g glycine add dd H O to 1 liter 48 mM Tris 39 mM glycine 20 v v methanol pH 9 2 Mix 5 82 g Tris and 2 93 g glycine in ddH O add 200 ml methanol Bring to 1 liter with ddH O 48 mM Tris 39 mM glycine pH 9 2 Mix 5 82 g Tris and 2 93 g glycine Add ddH 0 to 1 liter 10 mM NaHCO 3 mM NaCO 20 methanol pH 9 9 Mix 0 84 g NaHCO and 0 318 g NaCO in ddH O add 200 ml methanol Bring to 1 liter with ddH O 10 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 1 0x TBE Tris Borate EDTA pH 8 3 90 mM Tris Borate 1 mM EDTA 5x stock solution 54 g Tris base 27 5 boric acid 20 ml 0 5 M EDTA pH 8 0 Add 200 ml 5x stock to 800 ml ddH O to make 1 0x working solution 1x TAE Tris Acetate EDTA 40 mM Tris Acetate 1 mM EDTA 50x stock solution 242 g Tris base 57 1 ml glacial acetic acid 100 ml 0 5 M EDTA pH 8 0 1x working solution add 20 ml 50x stock to 980 ml ddH O Section 4 Strategies for Optimizing Electrophoretic Transfer 4 1 Optimizing Protein Transfer Generally quantitative elution of denatured high molecular weight proteins is difficult The f
12. 1 876 56 29 Belgium Bio Rad Laboratories S A N V Begoniastraat 5 9810 Nazareth Eke Phone 09 385 55 11 Fax 09 385 65 54 Canada Bio Rad Laboratories Canada Ltd 5671 McAdam Road Mississauga Ontario L4Z 1N9 Phone 905 712 2771 Fax 905 712 2990 China Bio Rad Laboratories 14 Zhi Chun Road Hai Dian District Beijing 100088 Phone 01 2046622 Fax 01 2051876 Denmark Bio Rad Laboratories Symbion Science Park Fruebjergvej 3 DK 2100 Copenhagen Phone 39 17 9947 Fax 39 27 1698 Finland Bio Rad Laboratories Business Center L nsikeskus Pihat rma 1A SF 02240 Espoo Phone 90 804 2200 Fax 90 804 1100 France Bio Rad S A 94 96 rue Victor Hugo B P 220 94 203 Ivry Sur Seine Cedex Phone 1 49 60 68 34 Fax 1 46 71 24 67 Germany Bio Rad Laboratories GmbH HeidemannstraBe 164 D 80939 Minchen Postfach 450133 D 80901 M nchen Phone 089 31884 0 Fax 089 31884 100 India Bio Rad Laboratories C 248 Defence Colony New Delhi 110 024 Phone 91 11 461 0103 Fax 91 11 461 0765 Italy Bio Rad Laboratories Sr Via Cellini 18 A 20090 Segrate Milano Phone 02 21609 1 Fax 02 21609 399 J apan Nippon Bio Rad Laboratories 7 18 Higashi Nippori 5 Chome Arakawa ku Tokyo 116 e Phone 03 581 1 6270 Fax 03 5811 6272 The Netherlands Bio Rad Laboratories B V Fokkerstraat 10 3905 KV Veenendaal Phone 0318 540666 Fax 0318 542216 New Zealand Bio Rad Laboratories Pty Ltd P O Box 100 051 North Shore Mail Cent
13. TOCK ITEMS www artisantg com WeBuyEquipment 7 EQUIPMENT DEMOS HUNDREDS OF InstraV ea REMOTE INSPECTION LOOKING FOR MORE INFORMATION MANUFACTURERS Remotely inspect equipment before purchasing with Visit us on the web at www artisantg com 7 for more our interactive website at www instraview com information on price quotations drivers technical LEASING MONTHLY specifications manuals and documentation RENTALS ITAR CERTIFIED EE Contact us 888 88 SOURCE sales artisantg com www artisantg com
14. ade sec ondary antibody 2 Monoclonal antibodies may react non specifically with SDS denatured proteins e Compare the binding of other monoclonal or polyclonal antibodies e Blot native proteins as a comparison 3 Nonsense interactions are occurring due to ionic associations For example avidin a glyco sylated protein may bind to more acidic proteins on blots e Increase the ionic strength of the incubation buffers Increase the number duration or stringency of the washes Include progressively stronger detergents in the washes e g SDS is stronger than NP 40 which is stronger than Tween 20 Include Tween 20 in the antibody dilution buffers to reduce nonspecific binding No reaction or weak signal 1 The sample load was insufficient e Increase the amount of protein applied Concentration of the sample prior to loading may be necessary Use a more sensitive assay system 2 Insufficient antigen binding to the membrane is occurring e Stain the gel after transfer or use prestained or Kaleidoscope standards to assess transfer efficiency See the previous section for suggestions on improving transfer related problems 3 Primary or secondary antibodies may be inactive or non saturating e Store the reagents at recommended conditions Avoid repeated freeze thaw cycles bacterial contamination or heat inactivation e Detergents may affect the activity of some antibodies Eliminate them from the assay except for the wash aft
15. d initial coupling and higher repetitive yields In addition PVDF membrane exhibits better binding efficiency of blotted material in the presence of SDS in the transfer buffer PVDF must first be wetted in 100 MeOH but can then be used in buffer which does not contain MeOH 5 2 DNA and RNA Blotting Membranes Zeta Probe Nylon Membrane Nitrocellulose is not a suitable medium for electrophoretic transfer of nucleic acids as high concentrations of salt 10 x SSC are required for efficient binding 3 Molecules lt 500 bp are not bound at all even at high salt Low resistance results when an electric current is passed through a solution of high salt This causes potentially damaging high currents and power at very low voltages Since V cm is the eluting force inefficient transfer occurs under condi tions required for proper binding Zeta Probe membrane allows efficient binding of all sizes of single stranded DNA and RNA in the presence of low ionic strength buffers Zeta Probe membrane is an ideal alternative to nitrocellulose for the analysis of nucleic acids Binding is more stable through post transfer washes and reprobing may be performed as many as 10 times 14 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Table 5 1 Guide to Protein Blotting Membranes A variety of blotting membranes is available for immunoblotting each with particular advan tages depending on the needs
16. e current readings e Alterations in buffer make up i e addition of SDS or changes in ion concentration due to addition of acid or base to adjust the pH of the buffers e Gel pH ionic strength and percentage of acrylamide especially if the gel has not been properly equilibrated e Number of gels current increases slightly as the number of gels increases e Volume of buffer current increases when volume increases e Platinum mass current increases when mass increases e Transfer temperature current increases when temperature increases e Time in transfer at which reading was taken current normally increases as the buffer ing capacity diminishes with progress of the run Pre equilibration of gels All electrophoresis gels should be pre equilibrated in transfer buffer prior to electrophoretic transfer Pre equilibration will facilitate the removal of contaminating electrophoresis buffer salts and neutralization salts salts resulting from the denaturation of nucleic acids prior to transfer If the salts are not removed they will increase the conductivity of the transfer buffer and the amount of heat generated during the transfer Also low percentage gels lt 12 will shrink in methanol buffers Equilibration allows the gel to adjust to its final size prior to elec trophoretic transfer Current limits The PowerPac 200 Power Supply is capable of a 200 watt output Unless a current limit is set uncontrolled conductivity change
17. e should develop within 15 min utes If color fails to develop within 25 minutes the conjugate solution is suspect Repeat the procedure with a freshly prepared dilution of conjugate 3 Activity test for the first antibody solution e Use an ELISA RID Ouchterlony immunodiffusion or precipitation test to deter mine reactivity of the antibody with the antigen If possible repeat the assay proce dure with several dilutions of first antibody solution 6 3 Total Protein Detection Colloidal Gold Total Protein Stain high background 1 The blocking step is insufficient or omitted e Block with 0 3 Tween 20 in TBS using three washes of 20 minutes each 2 The membrane used is not compatible with this stain e Positively charged nylon membranes cannot be used with Colloidal Gold stain Use the Biotin Blot Total Protein Detection Kit instead 3 Contamination of the membrane occurred at a previous step 6 electrophoresis or transfer e Discard and remake the gel and transfer solutions e Replace or thoroughly clean contaminated fiber pads 4 Excessive amounts of protein are loaded on the gel or too much SDS is used in the trans fer buffer Proteins can pass through the membrane without binding and recirculate through a tank blotting system e Reduce the amount of protein on the gel or SDS in the transfer buffer Add a second sheet of membrane to bind excess protein 5 Colloidal gold stain solution is contaminated 20 Artisan T
18. echnology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com e The stain is a reusable reagent Be sure to use a separate clean plastic container to store previously used reagent in the refrigerator Discard any reagent that has viscous sediment at the bottom of the bottle If the solution does not have a dark burgundy color but is a light blue the stain was contaminated with buffer salts Buffer salts will react with the gold sol causing non specific precipitation of the reagent onto the membrane Discard this solution Colloidal Gold Total Protein Stain low sensitivity 1 Increase the incubation time for detection of low level signals e Overnight incubations are possible although background staining can increase 2 Transfer is incomplete e See poor transfer for suggestions on how to enhance transfer efficiency 3 Stain is exhausted as evidenced by the loss of the dark burgundy color and longer stain ing times e Discard the reagent 4 Buffer salt contamination has occurred The solution will be light blue instead of dark bur gundy e Discard the reagent 5 The sample load may be too low for the reagent to detect e Use the Gold Enhancement Kit for detection levels as low as 10 pg of protein per band Biotin Blot Total Protein Detection high background 1 Blocking conditions are insufficient e Match the blocker to the membrane Nylon membranes require the addition of 1 methy 2 py
19. ees 18 6 3 Total Protein Detection 0 ee eeceeesseeeeeseescesceeeeesecsecsecsecsessecsessessesseesesseseeeeenees 20 Section 7 Product Information A 23 S ction References ereire ee deed deed de Eeer gee 23 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 1 Introduction Blotting was first performed by Southern in 1975 with the transfer of DNA from agarose gels to nitrocellulose membranes Since that time blotting has been applied to RNA and proteins gt P in both agarose and polyacrylamide gels To circumvent the inefficiencies observed in various capillary transfers electric current has been adopted for eluting proteins from polyacrylamide gels as first described by Towbin et al in 1979 The use of electrophoretic transfer has also been applied to DNA and RNA blotting 3 Numerous publications have dealt with the topic of protein electrophoretic transfer techniques 5 There have also been reviews summarizing the expanding literature being generated on electrophoretic blotting methodology 77 31 For a more comprehensive listing of references and applications refer to bulletin 1721 or in the US call Bio Rad s Technical Services Group in Hercules California toll free at 1 800 4BIORAD 1 800 424 6723 The Mini Trans Blot cell is one component of Bio Rad s Modular Mini Electrophoresis System This system includes the Mini PROTEAN II dual slab cell for runnin
20. er blocking e Ifthe antibody titer is too low optimize the concentration using a dot blot experiment e Increase the antibody incubation times 4 The enzyme conjugate is inactive or non saturating e Test the reagent for activity see below e Store the reagents at recommended conditions Avoid repeated freeze thaw cycles bacterial contamination or heat inactivation 19 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com e Sodium azide is a potent inhibitor of horseradish peroxidase Use Thimerosal as a bacteriostat e Impure water may cause inactivation of the enzyme Use only distilled deionized water e Ifthe conjugate concentration is too low optimize using a dot blot experiment 5 Color development reagent is inactive e Test the reagent for activity see below and remake if necessary Tests for monitoring reagent activity 1 Activity test for the color development solution e Combine 1 0 ml of the color development solution with 10 ul of full strength second antibody conjugate The color reaction should develop immediately If color fails to develop within a few minutes the color development solution is inactive Make up a fresh working solution and repeat the color development assay 2 Activity test for the conjugate solution e Combine 1 0 ml of the color development solution tested above and 1 0 ml of the 1 3 000 dilution conjugate solution A light blue ting
21. esult ing in fixation of large molecular weight proteins within the gel matrix Use of PVDF membrane for SDS protein transfers eliminates the alcohol requirement and constitutes a logical strategy for analysis of high molecular weight or difficult to transfer proteins 7 PVDF must be wetted in 100 methanol but may then be used in buffer without menanol Limited protease treatment A protocol for protease digestion of protein during transfer has been published Efficient trans fer without loss of immunological reactivity was reported Alter membrane type As mentioned in 7 PVDF membrane allows transfer in the absence of alcohol Alter gel system If possible use non denaturing gradient pore gels for separation of proteins by molecular weight Isoelectric focusing gels or native gels may be considered if separation by molecu lar weight is not mandatory 12 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Enhance gel membrane contact Failure of molecules to bind efficiently to the membrane caused by poor gel membrane con tact is often confused with inefficient elution Poor contact is usually due to excess moisture in the gel membrane interface Proper technique and the use of a test tube or glass pipet as a rolling pin should assure good contact Proper selection of filter paper spacers will help assure good compression Gel and membrane equilibration in transfe
22. ffer chamber and lid Mini Trans Blot Cell Accessories 170 3931 Mini Gel Holder Cassette 170 3932 Filter Paper 7 5 x 10 5 cm 50 170 3933 Fiber Pads 8 x 11 cm 4 170 3934 Bio Ice Cooling Unit Section 8 References 1 Southern E M J Mol Biol 98 503 1975 2 Alwine J C Kemp D J Parker B A Reiser J Renart J Stark G R and Wahl G W Methods Enzymol 68 220 1979 Thomas P S Proc Nat Acad Sci 77 5201 1980 Seed B Nuc Acids Res 10 1799 1982 Renart J Reiser J and Stark G R Proc Nat Acad Sci 76 3116 1979 Bowen P Steinberg J Laemmli U K and Weintraub H Nuc Acids Res 8 1 1980 Towbin H Staehelin T and Gordon J Proc Nat Acad Sci 76 4350 1970 Bittner M Kupferer P and Morris C R Anal Biochem 102 459 1980 Stellwag E J and Dahlberg A E Nuc Acids Res 8 299 1980 Kutateladze T V Axelrod B D Gorbulev V G Belzhelarshaya S N and Vartikyan R M Anal Biochem 100 129 1979 Peudelhuber T L Ball D J Davis A H and Garrard W J Nuc Acids Res 10 1311 1982 Danner D B Anal Biochem 125 139 1982 Bio Rad Technical Bulletin 1110 Zeta Probe Blotting Membranes 1982 Holland L J and Wangh L H Nuc Acids Res 10 3283 1983 Syminton J Green M and Brackmann K Proc Nat Acad Sci 78 177 1981 Reiser J and Wardale J Eur J Biochem
23. g SDS PAGE electrophoresis gels the Mini Tube Cell for running first dimension IEF tube gels for 2 D applications and the Model 422 Electro Eluter for rapid efficient recovery of proteins and nucleic acids from electrophoresis gels The unique feature of this electrophoresis system is that the electrode modules are interchangeable After finishing one task remove the electrode module from the buffer tank insert a new electrode module add a new buffer and the next electrophoresis application can be performed The Mini Trans Blot module accommodates two cassettes for electrophoretic transfer of both gels generated by the Mini PROTEAN II cell The Mini Trans Blot module is useful for blotting either protein or nucleic acid samples from both agarose and acrylamide gels It is also capable of blotting isoelectric focusing gels run on horizontal electrophoresis cells or DNA and RNA gels from the Mini Sub submarine electrophoresis cell For applications where the gel is larger than 7 5 x 10 cm or when there are many gels to be transferred the larger standard Trans Blot cell catalog number 170 3910 or 170 3946 or the Trans Blot SD semi dry cell catalog number 170 3940 should be used The heart of the Mini Trans Blot cell is its electrode module This module has the capac ity to hold two gel cassettes between parallel electrodes only 4 cm apart The driving force for blotting applications is the voltage applied over the distance between the elec
24. he cell to transfer samples Bio Rad recommends that you first read these instructions carefully Then assem ble and disassemble the cell completely After these preliminary steps you should be ready to transfer a sample Wash Cell Before Use Bio Rad also recommends that all Mini Trans Blot electrophoretic transfer cell compo nents and accessories be cleaned with a suitable laboratory cleaner such as Bio Rad Cleaning Concentrate catalog number 161 0722 and rinsed thoroughly with distilled water before use Warranty Model Catalog Number Date of Delivery Warranty Period Serial Number Invoice Number Purchase Order Number Bio Rad Laboratories warrants the Mini Trans Blot electrophoretic transfer cell against defects in materials and workmanship for year If any defects occur in the instrument dur ing this warranty period Bio Rad Laboratories will repair or replace the defective parts free The following defects however are specifically excluded Defects caused by improper operation Zeit AS Repair or modification done by anyone other than Bio Rad Laboratories or an authorized agent Use of fittings or other spare parts supplied by anyone other than Bio Rad Laboratories Damage caused by accident or misuse Damage caused by disaster Dn w Corrosion due to use of improper solvent or sample For any inquiry or request for repair service contact Bio Rad Laboratories after con firm
25. ing buffer salts compete for the biotinylation reagents e Wash the membrane thoroughly in Borate Tween to remove any residual buffer salts from electrophoresis and transfer 5 Avidin HRP conjugate is inactive e Follow the activity test procedures to determine if the reagent is inactive 6 Color development solution is inactive e Follow the activity test procedures to determine if the reagent is inactive Anionic dyes high background 1 Destaining is insufficient e Increase the number and duration of washes with the destaining solution 2 Dye solution is too concentrated e Remake the solution 3 Nylon membranes are not compatible with anionic dyes e Use the Biotin Blot Protein Detection Kit Anionic dyes low sensitivity 1 Anionic dye stains do not detect protein bands below 100 ng e Use amore sensitive stain such as the Colloidal Gold stain or the Biotin Blot Protein Detection Kit e Increase the sample load to achieve the detection level of the anionic dye stains 22 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Section 7 Product Information Catalog Number Product Description Mini Trans Blot Cell 170 3930 Mini Trans Blot Electrophoretic Transfer Cell includes 2 Gel Holder cassettes modular electrode assembly Bio Ice cooling unit lower buffer chamber and lid with cables 170 3935 Mini Trans Blot Module same as 170 3930 without lower bu
26. ing the model and serial number of your instrument Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Table of Contents Section 1 Totrolugin cscs oegeEegeree Seege eet stdaciesnsiecntbesscecestsoecdaoncasedecnatettacwtsdeces 1 UR WE te Lee issehscces teers tava Hts e dae teh teas toes Tere then Sees 2 L Saey MEMU NONS stitch G tite tetea teicher tee 3 Section 2 Mini Trans Blot Cell Assembly and Preparation for Transfer 4 2 1 Mini Trans Blot Cell Description and Assembly AAA 4 2 2 Preparation for Blotting 220 25 cessesieecseceissestisiessieessisdesnonssnteevnnhiebateneieneae 5 23 Acidic Transfersincccncdsaacananaain cnaiacnaahacanahanaccandanaaaadd 8 Section3 Transfer Conditions 8 3 1 General Guide for Transfer Buffers and Running Conditions eee 8 3 2 Notes on Electrophoretic Transfer Conditions ceeesesessseeeeeseeseeeseseeeeeeseseeees 10 3 3 B ffer Her 11 Section 4 Strategies for Optimizing Electrophoretic Transfer 12 4 1 Optimizing Protein Transfer 11 12 4 2 Optimizing DNA and RNA Transfer 13 Section5 Choice of Blotting Membranes e 14 5 1 Protein Blotting Membranes 1 14 5 2 DNA and RNA Blotting Membranes 1 14 Section 6 Troubleshooting Guide 15 6 1 Electrophoretic Transfers ici ccccsccicckccisccei cack steidectssedscveccestecsidancessatecdesnscvaanentdanceevicess 15 6 2 Immumne Specific Detection ee eceesssececceeceeceeceececeececseeecececseseseeeeseeseen
27. ites are easily and rapidly blocked avoiding subsequent background problems No pre activation is required Low molecular weight proteins especially lt 20 000 daltons may be lost during post transfer wash es thus limiting detection sensitivity Smaller pore size nitrocellulose membrane 0 2 um has been shown to be effective in eliminating this Joss 77 Large proteins C 100 000 daltons denatured by SDS may transfer poorly due to the addition of alcohol to the transfer buffer Alcohol increases binding of SDS proteins to nitrocellulose but decreases pore sizes in the gel Elimination of alcohol from SDS protein transfers results in considerably diminished binding Adding SDS up to 0 1 to the transfer buffer increases the transfer efficiency of proteins but reduces the amount of binding to the membrane Also SDS increases the con ductivity of the buffer and the heat generated during transfer PVDF Membrane PVDF Polyvinylidene difluoride membrane is an ideal support for amino terminal sequencing amino acid analysis and immunoassays of blotted proteins PVDF retains proteins under extreme conditions of exposure to acidic or basic conditions and in the presence of 13 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com organic solvents Greater retention during sequencing manipulations enhances the likelihood of obtaining information from rare low abundance proteins by increase
28. ize 7 5 cm x 10 cm Buffer capacity With cooling unit 650 ml Without cooling unit 850 ml Cleaning Use mild soap and warm water to clean the electrodes cassettes and buffer tank Use spe cial care when cleaning the electrode cards or plate electrodes Avoid stretching or breaking the platinum wires Avoid scratching or marring the platinum plate Do not use abrasives or strong detergents The cathode plate stainless steel can be cleaned with a mild abrasive to remove salt that may be deposited during nor mal operation Rinse the fiber pads under hot water and then in distilled deionized water Chemical compatibility The Mini Trans Blot cell components are not compatible with chlorinated hydrocarbons e g chloroform aromatic hydrocarbons e g toluene benzene or acetone Use of organic solvents voids all warranties Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 1 2 Safety Instructions Power to the Mini Trans Blot cell is supplied by an external DC voltage power supply N This power supply must be ground isolated in such a way that the DC voltage output floats with respect to ground All of Bio Rad s power supplies meet this important safety require ment Regardless of which power supply is used the maximum specified operating parame ters for the cell are 150 VDC Maximum voltage limit 40 Watts Maximum power limit 50 C Maximum ambient temperature limit
29. l cause damage to the instrument Section 3 Transfer Conditions 3 1 General Guide for Transfer Buffers and Running Conditions Table 3 1 provide guidelines for power conditions using different buffers Power condi tions are provided for various run times Where multiple conditions are displayed the high er the voltage the less time required for the run Always use the Bio Ice cooling unit Table 3 1 Guide to Buffers and Running Conditions High Intensity Field Standard Field 4 cm electrode distance Buffer Overnight Transfer 1 Hour Transfer SDS PAGE Gels Buffer A or B or C Buffer A or B or C A 25 mM Tris pH 8 3 192 mM 30 V 100 V glycine with or without 90 mA 350 mA 20 MEOH and 025 0 1 SDS B 48 mM Tris pH 9 2 39 mM glycine with or without 20 MEOH and 025 0 1 SDS C 10 mM NaHCO 3 mM NaCO pH 9 9 with or without 20 MEOH and 025 0 1 SDS DNA and RNA TAE 20 mM Tris pH 7 8 10 mM 30 V 80 V sodium acetate 0 5 mM EDTA 100 mA 500 mA TBE 50 mM Tris pH 8 3 50 mM sodium borate 1 0 mM EDTA Native Gels 25 mM Tris pH 8 3 30 V 100 V 192 mM glycine No methanol 90 mA 350 mA Isoelectric Focusing Native Gels Basic Proteins Acid Urea Gels 0 7 acetic acid 30 V 100 V 100 mA 350 mA Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 2 Notes on Electrophoretic Transfer Conditions These variables will change total resistance and thus th
30. mentation Guaranteed 888 88 SOURCE www artisantg com 4 Close the cassette firmly being careful not to move the gel and filter paper sandwich Lock the cassette closed with the white latch 5 Place the cassette in module Repeat for the other cassette 6 Add the frozen Bio Ice cooling unit Place in tank and completely fill the tank with buffer Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 7 Add a standard stir bar to help maintain even buffer temperature and ion distribution in the tank Set the speed as fast as possible to keep ion distribution even 8 Put on the lid plug the cables into the power supply and run the blot Refer to Section 3 for run times and voltage settings with various buffers 9 Upon completion of the run disassemble the blotting sandwich and remove the mem brane for development Clean the cell fiber pads and cassettes with laboratory detergent and rinse well with deionized water Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 2 3 Acidic Transfers If transferring under acidic conditions switch the gel and membrane in the set up instruc tions This will place the membrane on the cathode side of the gel Under acidic conditions proteins will transfer in the opposite direction going toward the negative cathode Do not reverse the electrodes themselves This wil
31. nated methanol can result in increased transfer buffer conductivity as well as poor transfer of macromolecules Do not reuse trans fer buffers or dilute transfer buffers below recommended levels Reuse of transfer buffers is not advised since these buffers have most likely lost their ability to maintain a stable solution pH during transfer Dilution of transfer buffers below their recommended levels is also not advised since this will decrease their buffering capacity Voltage limits Do not increase voltage settings beyond those indicated in Tables 3 1 3 4 for overnight oper ation Buffer conductivity must be close to the current listed and a current limit should be set on the power supply If overnight transfers at low voltages are ineffective for your application and higher voltages are necessary transfer times must also be decreased Failure to do so may result in a potential safety hazard 3 3 Buffer Formulation All formulas provided below are for a total volume of 1 liter of buffer Approximately 500 ml of buffer are required for the Mini Trans Blot cell Do not add acid or base to adjust pH of the following buffers Methanol should be analytical reagent grade as metallic contaminants in low grade methanol will plate on the electrodes Note Some pH electrodes will not perform a proper measurement for the pH of Tris buffers If the pH of the buffer is off check to make sure the electrode is designed to work with Tris buffers If
32. nductivity may be too high resulting in excessive power delivered to the cell See the power guide lines for specific applications in Section 3 16 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 A The membrane is not properly wet or has dried out e White spots on the nitrocellulose membrane indicate dry areas where protein will not bind If wetting does not occur immediately by immersion of the sheet in transfer buffer heat distilled water until just under the boiling point and soak the membrane until completely wet Equilibrate in transfer buffer until ready for use e Because of the hydrophobic nature of PVDF the membrane must be prewet in methanol prior to equilibration in aqueous transfer buffer Follow the directions in the product insert The gel electrophoresis may be at fault e Artifacts of electrophoresis may be produced by poor polymerization inappropriate running conditions contaminated buffers sample overload etc Gel cassette pattern transferred to blot 1 Contaminated or thin fiber pads are used e Replace the fiber pads or thoroughly clean the contaminated pads Excessive amounts of protein were loaded on the gel or too much SDS was used in the transfer buffer Proteins can pass through the membrane without binding and recirculate through the tank blotting system e Reduce the amount of protein on the gel and SDS in the transfer buffer
33. ollowing tactics alone or in combination will increase transfer efficiency Vary gel composition Gradient gels are often more effective than single gel concentrations for elution of a wide range of molecular weight proteins Lower the total monomer to create a more porous gel Increase or decrease the percentage of crosslinker A 5 26 C gel will contain the small est pore size of all gels no matter what the concentration of acrylamide An increase or decrease in C will make gels more porous with little loss in resolution C grams bis x 100 grams bis grams acrylamide Increase transfer time An initial control should be performed to determine the time required for complete transfer Times may vary from as little as 30 minutes to as long as overnight Remember all overnight applications should be performed at 30 volts to minimize heating problems Increase the power Initial controls should be performed to evaluate the efficiency of increasing the V cm as well as its effects on the temperature of transfer The temperature increase may change buffer resistance and subsequent power delivered as well as the state of protein denaturation thus affecting transfer efficiency 11 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Reduce buffer strength Dilution of transfer buffer results in lower current at any given voltage This will allow the use of higher voltages
34. oni J M in Methods of Biochemical Analysis Vol 33 Glick D ed Wiley New York in press 34 Bjerrum O J and Schafer Nielsen C Analytical Electrophoresis M J Dunn ed VCH Weinheim p 315 35 Dunn S D Anal Biochem 157 144 1986 36 Zeta Probe Instruction Manual Bio Rad Laboratories 1988 37 Polvino W J Saravis C A Sampson C E and Cook R B Electrophoresis 4 368 1983 39 Bio Rad Laboratories Biotin Blot Total Protein Stain Instruction Manual 1985 40 LaRochelle W J and Froehner S C J Immunol Meth 92 65 1986 41 Szewcyzyk B and Kozloff L M Anal Biochem 150 403 1985 42 Perides G Plagens U and Traub P Anal Biochem 152 94 1986 H H Scotch Brite is a registered trademark of 3M Company Gel Bond is a trademark of FMC Mylar is a registered trademark of E I DuPont de Nemours Co Coomassie is a trademark of ICI 24 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Life Science Group 2000 Alfred Nobel Drive Hercules California 94547 Telephone 510 741 1000 Fax 510 741 5800 Bio Rad Laboratories Australia Bio Rad Laboratories Pty Limited Block Y Unit 1 Regents Park Industrial Estate 391 Park Road Regents Park NSW 2143 Phone 02 9414 2800 Fax 02 9914 2888 Austria Bio Rad Laboratories Ges m b H Auhofstrasse 78D 1130 Wien Phone 1 877 89 01 Fax
35. r buffer for 30 minutes to 1 hour prior to transfer will help prevent shrinking of either component during transfer and will eliminate reactants such as urea or SDS from the gel 4 2 Optimizing DNA and RNA Transfer Problems with elution of nucleic acids can be solved by altering the gel percentage It may be somewhat more difficult to quantitatively transfer large amounts of DNA used in genomic blots The following tactics should be considered for optimizing elution in such transfers Alter gel composition Lower total monomer or crosslinker for polyacrylamide gels Lower agarose This allows better elution of high molecular weight DNA Alter DNA denaturants It has been found that glyoxal denaturation allows more efficient elution of DNA than NaOH Boiling polyacrylamide gels to denature DNA has also been found to give excellent results 1 Base denaturation often causes polyacrylamide gels to weaken and stick to blotting membranes Section 5 Choice of Blotting Membranes 5 1 Protein Blotting Membranes Nitrocellulose Membrane Nitrocellulose membranes have been used extensively for protein binding and detection 7732427 They can be easily stained for total protein by a dye stain Amido Black Coomassie Blue Ponceau S Fast Green FCF etc A7 or the more sensitive Colloidal Gold Total Protein Stain and also allow either RIA FIA or EIA Nitrocellulose has a high bind ing capacity of 80 100 pg cm Nonspecific protein binding s
36. re Auckland 10 e Phone 09 443 3099 Fax 09 443 3097 Pacific Bio Rad Laboratories Unit 1111 11 F New Kowloon Plaza 38 Tai Kok Tsui Road Tai Kok Tsui Kowloon Hong Kong Phone 7893300 Fax 7891257 Singapore Bio Rad Laboratories Singapore Ltd 221 Henderson Rd 05 19 Henderson Building Singapore 0315 Phone 65 272 9877 Fax 65 273 4835 Spain Bio Rad Laboratories S A Avda Valdelaparra 3 Pol Ind Alcobendas E 28100 Alcobendas Madrid Phone 91 661 70 85 Fax 91 661 96 98 Sweden Bio Rad Laboratories AB Gardsvagen 7D Box 1276 S 171 24 Solna Phone 46 0 8 735 83 00 Fax 46 0 8 735 54 60 Switzerland Bio Rad Laboratories AG Kanalstrasse 17 Postfach CH 8152 Glattbrugg Phone 01 809 55 55 Fax 01 809 55 00 United Kingdom Bio Rad Laboratories Ltd Bio Rad House Maylands Avenue Hemel Hempstead Herts HP2 7TD Free Phone 0800 181134 Fax 01442 259118 SIG 020996 Printed in USA M1703930 Rev E Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com A rtisan Artisan Technology Group is your source for quality Fra op new and certified used pre owned equipment FAST SHIPPING AND SERVICE CENTER REPAIRS WE BUY USED EQUIPMENT DELIVERY Experienced engineers and technicians on staff Sell your excess underutilized and idle used equipment TENS OF THOUSANDS OF at our full service in house repair center We also offer credit for buy backs and trade ins IN S
37. rrolidinone MPO to several solutions Consult the Biotin Blot manual for specific details 2 Membrane is left in color development solution too long e Remove the membrane from the color development solution when the signal is appar ent and the background has not developed Transfer the blot to distilled water immedi ately to stop the development 3 Excessive amounts of protein are loaded on the gel or too much SDS is used in the trans fer buffer Proteins can pass through the membrane without binding and recirculate through a tank blotting system e Reduce the amount of protein on the gel or SDS in the transfer buffer Add a second sheet of membrane to bind excess protein Biotin Blot Total Protein Detection no reaction or weak color development 1 Transfer is incomplete e See Poor Transfer for suggestions on how to enhance transfer efficiency 2 The sample load may be too low for the reagents to detect e Increase the amount of protein loaded on the gel 21 Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 3 NHS biotin solution is inactivated e NHS biotin hydrolyzes in aqueous solutions Equilibrate the reagent vial to room temperature before opening to prevent condensation of water inside the container Use a sterile syringe to remove reagent to prevent contamination e Add the NHS biotin reagent to the Borate Tween solution just prior to use 4 Amine contain
38. s may result in full power being delivered to the Mini Trans Blot cell The gel holders may warp and the transfer buffer may boil and evaporate further increasing conductivity This would result in a potential safety hazard Refer to the PowerPac 200 Power Supply Instruction Manual for setting current limits and run times Polarity of transfer Do not reverse polarity with the plate electrodes Use of a stir bar during transfer For all blotting applications a stir bar must be placed inside the Mini Trans Blot cell so that the transfer buffer is stirred during the course of the experiment This will help to maintain uni form conductivity and temperature during electrophoretic transfer Failure to properly control transfer buffer temperature results in poor transfer of macromolecules and poses a potential safety hazard Transfer buffer pH Do not adjust the pH of transfer buffers unless specifically indicated Adjustments of the pH of transfer buffers when not indicated will result in increased buffer conductivity This is manifested by a higher than expected initial current output and a decreased resistance It is rec ommended that the buffer conductivity and resistance be checked with the PowerPac 200 Power Supply before starting each transfer Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com Transfer buffer recommendations Use only high quality reagent grade methanol Contami
39. trodes This short 4 cm electrode distance allows generation of higher driving forces to produce efficient protein transfers A second feature of the electrode module is that it is offset to accommo date a Bio Ice cooling unit The cooling unit which is completely contained within the Mini Trans Blot cell absorbs the Joule heat generated during rapid electrophoretic transfers The advantages of having an internal cooling unit include elimination of an expensive external cooling bath and avoidance of cumbersome cooling tubing that always seems to be in the way Other features of the Mini Trans Blot cell include latches on the gel holder cassettes for easy handling color coordinated cassettes and electrodes to insure proper orientation of the gel during transfer and an efficient design which simplifies insertion and removal of the cassettes from the electrode assembly The result of these features is an electrophoretic transfer system which is easy to use and which produces excellent blotting results Artisan Technology Group Quality Instrumentation Guaranteed 888 88 SOURCE www artisantg com 1 1 Specifications Construction Electrode module Molded polysulfone Gel holder cassettes Molded polycarbonate Electrodes Platinum wire 0 254 mm diameter Buffer chamber and lid Molded polycarbonate Cooling unit Polyethylene Overall dimensions Mini Trans Blot cell 16 cm L x 12 cm W x 18 cm H Gel holder dimensions 10 cm x 11 cm Maximum gel s
40. without excessive heating Vary buffer type and pH Maximize charge to mass ratio It appears that alcohols present in SDS transfer buffer strip SDS from proteins Basic proteins in Tris glycine methanol buffer at pH 8 3 may assume a state near isoelectric neutrality and thus transfer poorly For example lysozyme exhibits this behavior Buffers with pH of 9 5 to 10 0 have shown much better elution and binding characteristics for basic proteins such as lysozyme and histones Different buffer types at similar V cm may yield different efficiencies Generally Tris buffers allow more efficient transfer than acetate or phosphate buffers Add detergent Addition of 0 1 SDS detergent to Tris glycine methanol buffer has been reported to increase transfer efficiency SDS however increases relative current power and heating Also tem peratures below 10 C may precipitate the SDS so the starting buffer temperature will be higher SDS may also affect the antigenicity of some proteins SDS will aid in eluting the proteins from the gel but it may reduce the binding efficiency of those proteins to the nitro cellulose membrane Eliminate alcohol from the transfer buffer Alcohol in the transfer buffer improves binding of SDS proteins to nitrocellulose only Elimination of alcohol results in increased transfer efficiency but diminishes binding to nitro cellulose Transfer efficiency is increased because alcohol causes gel pores to contract r
Download Pdf Manuals
Related Search