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- Proteins and Peptides
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BATCH NUMBER 111359 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No bands were detected. SAMPLE 30 micro g HeLa whole cell extract. PRIMARY ANTIBODY ab4143 Rabbit 1:100 in 5% milk in PBST for 1 hour. wash 3 times for 5 minutes in PBST with gentle rocking. DETECTION METHOD Enhanced chemiluminescense (Pierce) POSITIVE AND NEGATIVE CONTROLS USED HeLa cell extracts are positive for PKC delta. No negative control used. Other lanes with the same lysate were probed for different antigens with the same secondary antibodies, so, the sample was OK, the gel worked, the transfer worked, and the secondary worked. ANTIBODY STORAGE CONDITIONS The antibody is stored at -20 C. It has been at 4 C for about a week. SAMPLE PREPARATION RIPA buffer, followed by immediate protein concentration determination, dilution and addition of reducing sample buffer then 5 minutes at 95 C (<2 hours from sample collection to sample buffer, on ice the whole time). AMOUNT OF PROTEIN LOADED 30 micro g ELECTROPHORESIS/GEL CONDITIONS 4-15% gradient rready gel from bioRad TRANSFER AND BLOCKING CONDITIONS Transfer with biorad apparatus and buffers. Two hours at 45 volts, constant. Block in 5% milk in PBS+Tween for 1 hour. SECONDARY ANTIBODY [a competitor] anti rabbit IgG 1:2000 in 2.5% milk in PBST. Wash 3X 5 minutes in PBST HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? None, that is why my results were inconclusive, I'll update my review as I try more conditions. ADDITIONAL NOTES If you have any suggestions, I'd appreciate them. |
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ANSWER: |
Thank you for contacting us for technical support, I'm sorry to hear you are experiencing problems with ab4143. I am currently in touch with the laboratory to find out the recommended positive control and would like to recommend an overnight incubation at 4C with the primary antibody in TBST. In our experience too much milk can prevent the antibody from blocking so by reducing the blocking stage to 30min or 1hr prior to antibody addition this should help you a lot. As soon as I find out the recommended positive control I will let you know. Could you please let me know how you get on with the modifications suggested above, together we will then be able to update your Abreview to reflect your results, |
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