ReliaBLOT®
Contents
1. eee 8 Electrophoresis in ReliaBLOT SDS PAGE Gel see 8 Transfer to Nitrocellulose sss 9 Western BlOt ne sax med ORG PEERTO RC BEC AMA ha 10 Development eterno e De Ty dd E Sete c peti EES 10 Troubleshooting irte r e TREE ARES 12 Introduction The ReliaBLOT Western Blot Kit from Bethyl Laboratories conveniently provides the major reagents needed to perform standard western blot assays The kit provides enough reagents to run five mini gels and western blot assays The components provided for SDS PAGE and gel transfer are compatible with most mini gel electrophoresis and blotting modules that accept an external gel cassette size of 10 X 8 cm e g Bio Rad Mini PROTEAN Cell and Mini Trans Blot modules For western blot an HRP conjugated Anti rabbit secondary antibody is provided for the detection of primary antibodies made in rabbit hosts Kit Components The ReliaBLOT Kit components quantities and storage conditions are as follows Component Quantity Storage ReliaBLOT Precast SDS PAGE Gel 5 each 2 8 C Cassettes 4 8 4 12 4 20 gradient or 12 12 well 10 X 8 cm ReliaBLOT LDS Sample Buffer 4x 2 0 ml Room Temperature ReliaBLOT DTT Reducing Agent 0 15 g 2 8 C OR 209C after reconstitution ReliaBLOT Pre stained Protein Marker 0 1 ml 20 C long term OR 2 8 C up to 3 mos ReliaBLOT Running Buffer 20X 125 ml Room Temperatur2. PAGE Gels 4 896 10 x 10 cm 10 gels WB101 40812G ReliaBLOT SDS PAGE Gels 4 12 10 x 10cm 10 gels WB101 01212G ReliaBLOT SDS PAGE Gels 4 20 10 x 10 cm 10 gels WB101 42012G ReliaBLOT SDS PAGE Gels 12 10x 10cm 10 gels WB101 41212G ReliaBLOT SDS PAGE Gels 4 8 10 x 8 cm 10 gels WB102 40812G ReliaBLOT SDS PAGE Gels 4 12 10x 8cm 10 gels WB102 01212G ReliaBLOT SDS PAGE Gels 4 20 10x 8 cm 10 gels WB102 42012G ReliaBLOT SDS PAGE Gels 12 10x 8 cm 10 gels WB102 41212G ReliaBLOT LDS Buffer 4X 10 ml WB104 10 ReliaBLOT Running Buffer 20X 500 ml WB105 500 ReliaBLOT Transfer Buffer 10X 500 ml WB106 500 ReliaBLOT Nitrocellulose Membrane Filter Sandwiches 7 5 x 8 3 cm 20 pk WB107 20 ReliaBLOT DTT Reducing Agent 10X 1 ml WB108 ReliaBLOT Prestained Protein Marker 600 ul WB103 600 ReliaBLOT Chemiluminescent HRP Substrate 110 ml WB110 ReliaBLOT IP Western Blot Reagents 20 blots WB120 Goat anti Rabbit IgG h HRP 1 ml at 1 mg ml A120 101P Goat anti Mouse IgG h HRP 1 ml at 1 mg ml A90 116P 16
3. or on ice or smiling 2 Slow down the run by bands across gel lowering the voltage Warranty Products are warranted by Bethyl Laboratories Inc to meet stated product specifications and to conform to label descriptions when used handled and stored according to instructions Unless otherwise stated this warranty is limited to six months from date of sale Bethyl Laboratories sole liability for the product is limited to replacement of the product or refund of the purchase price Bethyl Laboratories products are supplied for research applications They are not intended for medicinal diagnostic or therapeutic use The products may not be resold modified for resale or used to manufacture commercial products without prior written approval from Bethyl Laboratories Inc 15 Related Products Description Size Catalog No ReliaBLOT Western Blot Kit 4 896 10 x 10 cm kit WB101 40812K ReliaBLOT Western Blot Kit 4 12 10 x 10 cm kit WB101 41212K ReliaBLOT Western Blot Kit 4 2096 10 x 10cm kit WB101 42012K ReliaBLOT Western Blot Kit 12 10x 10cm 1 kit WB101 01212K ReliaBLOT Western Blot Kit 4 896 10 x 8 cm kit WB102 40812K ReliaBLOT Western Blot Kit 4 12 10x 8cm 1 kit WB102 41212K ReliaBLOT Western Blot Kit 4 20 10x 8cm 1 kit WB102 42012K ReliaBLOT Western Blot Kit 12 10x 8cm 1 kit WB102 01212K ReliaBLOT SDS
4. 2 Problem Cause Solution No Signal continued Poor transfer of protein to membrane continued Small proteins gt 20 kDa may transfer through the membrane Shorten transfer time or re evaluate the gel percentage and buffer system used Large proteins gt 200 kDa may reguire an overnight transfer at low voltage 2 8 C Exceptionally large proteins gt 300 kDa may require the use of a tris acetate gel and buffer system Re evaluate the gel percentage and buffer system used Target is masked by the blocking solution Experiment with alternatve blocking buffers e g BSA Lower the percentage of milk e g 1 Block for less time Chemiluminescent detection Confirm that the working solution was made properly Use freshly made working solution Sodium azide is an inhibitor of HRP Do not use sodium azide as a preservative in buffers High membrane background or dirty blot Insufficient blocking 5 non fat dry milk in TBS or PBS with Tween20 0 05 works well to block membrane background Block membranes for at least 1 hr at room temperature Use blocking buffer as the diluent for the primary antibody Ensure good coverage of the membrane with blocking solution during blocking and antibody incubation Ensure that the dried milk is fully dissolved in the blocking buffer Blocking buffer should be fresh Primary antibody Decrease the concentrat
5. ReliaBLOT Western Blot Kit 10 x 8 cm 12 12 well Cat No WB102 01212K User Manual BETHYL LABORATORIES INC The Polyclonal Antibody Specialists www bethyl com 1 800 338 9579 Table of Contents IntrOdUctiOD a Ay ar e t WYD YD AG AD Wd By 3 Kit Components s nu i nu beet ter REA tantum e 3 Other Reagents Needed ones edet ette etie inte FUN 4 Component Specifications essere WELE xak n ke dey xelka sf enne 4 ReliaBLOT Precast SDS PAGE Gel Cassettes 4 ReliaBLOT LDS Sample Buffer 4X 4 J eee keke eke kek 4 ReliaBLOT DTT Reducing Agent 4 ReliaBLOT Pre stained Protein Marker o ReliaBLOT Running Buffer 20X c sccccccsccssessessesscscssesscscsscsucsesecsecsesecsncssees 5 ReliaBLOT Transfer Buffer 10X uu III I tenentes 6 ReliaBLOT Nitrocellulose Membrane Filter Sandwiches 6 ReliaBLOT HRP Conjugated Anti Rabbit Secondary Antibody 6 ReliaBLOT Chemiluminescent HRP Substrate e 6 Suggested Protocols for SDS PAGE and Western Blot 7 Buffer and Reagent Preparation EL keke eke eee kek k e kek 7 ReliaBLOT Running Butetown AN 7 ReliaBLOT Transfer Buffer seen 7 ReliaBLOT DTT Reducing Agent sese 7 TBST uisi e dete ar ER PEPERIT ade 8 Blocking Buffer eite a eae ODER UR Re eR 8 Preparation of Protein Lysate Sample
6. e ReliaBLOT Transfer Buffer 10X 500 ml Room Temperature ReliaBLOT Nitrocellulose 5 each Room Temperature Membrane Filter Sandwiches 0 2 um ReliaBLOT HRP conjugated Anti 0 05 ml 2 8 C rabbit Secondary Antibody ReliaBLOT Chemiluminescent HRP 6 0 ml 2 8 C Substrate A and B each Other Reagents Needed o Methanol Primary Antibodies made in rabbit hosts Blocking Buffer e g non fat dried milk or BSA in TBS with Tween Wash Buffer Tris Buffered Saline with Tween TBST Ultrapure Water Distilled or Deionized Water ooooo Component Specifications ReliaBLOT Precast SDS PAGE Gel Cassettes External size of 10 cm W X 8 cm L X 1 cm gel matrix thickness Full length resolving gradient gel 4 8 4 12 4 20 or 12 Red stacking gel for easy well identification High capacity 12 well load up to 25 ul per well ReliaBLOT LDS Sample Buffer 4X ReliaBLOT LDS sample buffer 4X is formulated according to the table below 4X Tris HCL 424 mM Tris base 564 mM LDS 8 96 Glycerol 4096 EDTA 2 04 mM Coomassie Brilliant 0 88 mM Blue G250 Phenol Red 0 72 mM ReliaBLOT DTT Reducing Agent e Used as a reducing agent for preparation of protein samples for SDS PAGE e Final concentration after reconstitution is 1 0 M Dithiothreitol 10X solution ReliaBLOT Pre stained Protein Marker 10 recombinant proteins with apparent molecular weights ran
7. ed The recommended range for dilution is 1 10 000 to 1 20 000 Incubate the membrane in diluted HRP conjugated Anti rabbit Secondary Antibody for 60 minutes on a rocker platform Wash as directed in step 5 Development 1 Make a Working Solution of the Chemiluminescent Substrate by mixing 1 ml of component A and 1 ml of component B Two mls of Working Solution will be needed for each membrane Use clean serological pipettes to pipette the components into a 15 ml conical tube Using forceps remove the membrane from the last wash and blot the edge on a paper towel to remove excess wash buffer and place the membrane on a clean surface 10 Pipette 2 ml of the activated substrate solution onto the entire surface of the membrane and incubate at room temperature for 5 minutes Using forceps lift the membrane and drain off substrate Blot the edge of the membrane on a paper towel to remove excess substrate Place the membrane in plastic membrane protector e g plastic film wrap or a page protector Smooth out bubbles between the membrane and the plastic protector Expose the membrane to film or a charged coupled device CCD camera Exposure times will vary in length and will need to be empirically determined 11 Troubleshooting Problem Cause Solution No Signal The primary antibody may not be compatible with the secondary antibody provided in the kit Primary antibodies must be made in rabbit ho
8. ging from 7 6 kDa to 195 kDa The 28 kDa and 71 kDa markers appear orange for easy identification The marker is ready to load no need to boil The recommended load volume is 5 8 ul of the protein marker per well 195 195 142 Rs 142 342 7 ww 96 195 96 48 m a S 48 w 142 n ss 3 96 i 28 28 22 S 22 71 33 124 12 48 10 e ja 104 x 221 2E 4 896 4 12 4 20 12 The average apparent molecular weights kDa for the ReliaBLOT Pre stained Protein Markers in the ReliaBLOT SDS PAGE Tris Glycine system are shown 8 ul of ReliaBLOT Pre stained Protein Marker was loaded on the indicated ReliaBLOT SDS gel electrophoresed for 1 hour at 150 volts and transferred to nitrocellulose for 2 hours at 20 volts ReliaBLOT Running Buffer 20X ReliaBLOT Running Buffer 20X is formulated according to the table below 20X Tricine free base 0 8 M Tris free base 12M SDS 2 096 Sodium Bisulfite 50 mM ReliaBLOT Transfer Buffer 10X ReliaBLOT Transfer Buffer 10X is formulated according to the table below 10X Glycine 1 92 M Tris free base 0 25 M SDS 1 ReliaBLOT Nitrocellulose Membrane Filter Sandwiches 100 nitrocellulose membrane Pre cut and assembled into a membrane and filter sandwich 0 2 um pore size 8 3 X 7 3 cm dimensions ReliaBLOT HRP Conjugated Anti Rabbit Secondary Antibody HRP horseradish peroxidase conj
9. ion increase the dilution of the primary antibody Titrate the primary antibody to empirically determine the 13 Problem Cause Solution High membrane background or dirty blot continued Primary antibody continued optimal antibody dilution to achieve the best signal noise ratio The nature of some primary antibodies may always result in slight membrane background The stock solution of the primary or secondary antibodies contains aggregates Microfuge the antibodies at 14 000 X G for 10 minutes at 47C Insufficient washing After primary and secondary antibody incubation perform at least three 10 minute washes Include detergent 0 0596 Tween20 in the TBS or PBS wash buffer Increase number of washes Multiple bands or lane background Insufficient blocking Primary antibody 5 non fat dry milk in TBS or PBS with Tween20 0 05 works well to block non specific bands and lane background Block membranes for at least 1 hr at room temperature Use blocking buffer as the diluent for the primary antibody Decrease the concentration increase the dilution of the primary antibody Titrate the primary antibody to empirically determine the optimal antibody to achieve the best signal noise ratio Use affinity purified antibody Secondary antibody Decrease the concentration increase the dilution of the secondary antibody Incubate seconda
10. o Rad Mini Trans Blot modules 10X Transfer Buffer 100 ml Methanol 200 ml Distilled Water 700 ml Total volume 1000 ml Chill to 2 8 C before use ReliaBLOT DTT Reducing Agent Reconstitute the DTT by adding 1 0 ml of ultrapure water Aliquot 100 pl each into microcentrifuge tubes and store at 20 C TBST Tris free base 6 1g 50 mM NaCl 8 1g 138 mM Tween 20 500 ul 0 05 96 Distilled water to 1 0L Adjust pH to 8 0 with HCL Store at 4 25 C Blocking Buffer NOTE Blocking buffer should be made fresh and dissolved well Carnation non fat dry milk 25g TBST 50 ml Total volume 50 ml Preparation of Protein Lysate Sample For each well sample volume should not exceed 25 u1 The mass of sample reguired for detection by western blot should be empirically determined Typically 10 to 50 ug of cell lysate in sample buffer is loaded per well 1 2 ON For each sample aliquot 10 to 50 ug of cell lysate in a sterile microfuge tube 5 25 ul Add 4X LDS sample buffer to the sample to achieve a 1X concentration of LDS sample buffer Add 10X DTT to achieve a final concentration of 1X DTT Mix well Heat samples at 95 C for 5 minutes Quick spin condensate if needed Load immediately on ReliaBLOT SDS PAGE gel as described below Electrophoresis in ReliaBLOT SDS PAGE Gel 1 2 3 Cut open the package that contains the gel cassette and drain away the buffer Rinse the wells with distilled wa
11. re 1 Figure 1 Assembly of the Gel Sandwich Proteins will migrate toward the anode therefore in the sandwich the membrane should be closest to the anode Di Place the cassette into the transfer module Fill the tank to the appropriate level with cold 1X Transfer Buffer Transfer conditions will depend on the type of blotting module used Refer to the manufacturer s recommendations for transfer running time and voltage When the transfer is complete remove the membrane from the blotting module sandwich and place the membrane in a dish of blocking buffer 590 non fat dry milk in TBST the buffer should sufficiently cover the membrane 10 Discard the filter paper Western Blot mn Incubate the membrane in blocking buffer for 1 hour on a rocking platform shaker Dilute the primary antibody in 15 ml of blocking buffer 590 non fat dry milk in TBST For best results the optimal dilution of antibody should be empirically determined Pour off the blocking buffer from the membrane and replace with the diluted primary antibody mixture Incubate the membrane in diluted primary antibody for two hours to overnight with gentle rocking at room temperature Wash the membrane three times 10 minutes each time in TBST Dilute the HRP conjugated Anti rabbit Secondary Antibody in 15 ml of 590 non fat dry milk in TBST For best results the optimal concentration of the secondary HRP conjugated antibody should be empirically determin
12. ry antibody for 1 hour Titrate the secondary antibody to empirically determine the optimal antibody dilution to achieve the best signal noise ratio 14 Problem Cause Solution Multiple bands Too much lysate protein sample Empirically determine the optimal 5r lane loaded into the lane amount of lysate to load per lane Or Man e to achieve the best signal to background noise ratio continued The lysate is degraded 1 Store lysates and protein samples at 80 C 2 Avoid multiple freeze thawing 3 Keep protein samples on ice Cross reacting proteins Primary antibodies may cross react with off target proteins even under optimal conditions Modified proteins The target protein may be present in multiple modified forms e g phosphorylation ubiquitination glycosylation or as different splice variants or isoforms Protein multimers The protein target may form multimers Boil samples in SDS or LDS sample buffer before loading Ghost bands Primary antibody concentration Titrate the primary antibody to too high empirically determine the optimal antibody dilution to achieve the best signal noise ratio reverse white bands Secondary antibody concentration Titrate the secondary antibody to too high empirically determine the optimal antibody dilution to achieve the best signal noise ratio Uneven bands Gel was run too hot or too fast 1 Run the gel in the cold room omiling
13. sts Primary antibody is too dilute 1 Increase concentration lower the dilution of the primary antibody 2 Titrate the primary antibody to empirically determine optimal antibody dilution to achieve the best signal noise ratio Insufficient binding time Incubate primary antibody overnight at 2 8 C or room temperature Insufficient antigen Load at least 20 50 ug of protein The lysate protein sample is degraded 1 Store lysates and protein samples at 80 C 2 Avoid multiple freeze thawing 3 Keep protein samples on ice 4 _ Check lysate integrity by probing the blot with a control antibody e g anti actin or staining the membrane with Ponceau S Inadequate expression of the protein target in the lysate sample 1 Use a positive control lysate in which the endogenous target is known to be expressed at relatively abundant levels 2 Enrich the target by isolating nuclear membrane or mitochondrial extracts 3 Enrich the target by performing an immunoprecipitation 4 Examine the literature for treatments that may induce endogenous expression of the target Poor transfer of protein to membrane 1 Check that all of the colored markers of the protein standard have been transferred to the membrane 2 Monitor transfer efficiency by staining the gel with Coomassie blue or staining the membrane with Ponceau S 3 Useonly fresh transfer buffer 1
14. ter Place the gels on the buffer core and assemble the electrophoresis cell according to the manufacturer s directions e g Bio Rad Mini PROTEAN Cell modules 4 Fill the inner core chamber with fresh 1X running buffer to cover the sample wells about 125 ml If there are no leaks fill the outer chamber with the remaining running buffer 5 Using a pipette 1 ml volume flush the wells using the 1x running buffer from the inner chamber 6 Load the prepared samples using a Hamilton syringe or a pipettor fitted with gel loading tips 7 Run the gels at 150V until the dye front reaches the bottom of the gel approximately 60 minutes Transfer to Nitrocellulose 1 Use gloves and forceps to handle the nitrocellulose membranes For each gel to be transferred remove and separate a membrane filter paper sandwich from the blue interleaf paper Discard the blue interleaf paper 2 Inashallow tray pre wet the nitrocellulose membrane and blotting filter paper in 1X Transfer Buffer for at least 5 minutes 3 Inashallow tray soak blotting pads in 1X Transfer Buffer 4 Open the gel cassette by inserting a small metal spatula or gel knife into the gap between the plates and gently twisting the plates apart The gel will stick to one plate 5 Note the orientation of the gel and assemble the blotting sandwich as described in the manufacturer s instructions for the blotting module e g Bio Rad Mini Trans Blot module or according to figu
15. ugated goat immunoglobulin G IgG protein 1mg ml Supplied in phosphate buffered saline PBS containing 0 2 BSA and 0 1 Pro Clean 400 For use with primary antibodies made in rabbit Reacts specifically with rabbit IgG and with light chains common to other rabbit immunoglobulins Recommended dilutions for western blot and detection by chemiluminescence are in the range of 1 10 000 to 1 20 000 ReliaBLOT Chemiluminescent HRP Substrate A two component enhanced chemiluminescent substrate for detecting HRP on immunoblots components A and B Working Solution is prepared by mixing equal parts of component A and B Only 2 ml of Working Solution needed per membrane Working Solution is stable for 24 hours at room temperature After incubation of blot with Working Solution chemiluminescent signal may continue for up to 8 hours but will decrease with time Suggested Protocols for SDS PAGE and Western Blot Buffer and Reagent Preparation ReliaBLOT Running Buffer About 300 ml of Running Buffer is needed to run one gel or a pair of gels in a mini gel system e g Bio Rad Mini PROTEAN Cell modules 20X Running Buffer 15 ml Distilled water 285 ml Total volume 300 ml Store for up to 1 week at 2 8 C ReliaBLOT T ransfer Buffer NOTE It is recommended to prepare the 1X Transfer Buffer the same day of the transfer Approximately 950 ml is needed to transfer one gel or a pair of gels in a tank system e g Bi
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