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ANTIBODY CODE ab7747 BATCH NUMBER not sure ORDER NUMBER not sure DESCRIPTION OF THE PROBLEM No signal detected by 2 users. SAMPLE Cell lysates collected from human prostate cancer cell lines PRIMARY ANTIBODY Abcam Ab7747: dilution of 100 microlitres in 15ml TBST + 5% marvel, incubation overnight at 4 degrees. Washes: 1x15 mins and 3x5 mins in TBST. These conditions have worked before. SECONDARY ANTIBODY anti-rabbit, 1:2000 dilution in TBST +5% marvel, incubated for 1 hour at room temp. Washes as before These conditions have worked before. DETECTION METHOD Have tried ECL, ECLPlus and Supersignal. None of these give a band at the correct size. POSITIVE AND NEGATIVE CONTROLS USED Samples that gave a positive result with a different batch of antibody. Cells treated with siRNA against IL8 as negative control ANTIBODY STORAGE CONDITIONS Aliquotted at -20 celcius SAMPLE PREPARATION RIPA with PI tablet + sodium orthovanadate ELB with PI tablet Samples mixed with loading buffer + mercaptoethanol & heated to 95 degrees for 5 minutes. AMOUNT OF PROTEIN LOADED 40 micrograms ELECTROPHORESIS/GEL CONDITIONS 20% reducing gel These conditions have worked before. TRANSFER AND BLOCKING CONDITIONS Transfer for 2 hours in transfer buffer containing 40% methanol. Membrane checked using Ponceau-s. Blocked in TBST +5% marvel for 2 hours. These conditions have worked before. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes ADDITIONAL NOTES I have tried the western 3 times and another user has tried twice. I have tried samples that gave a positive result with a previous batch of antibody. Different sample preparation buffers give no different result. e see a non-specific band arounf 40kDa (have seen this before) but no IL8 band.
Asked on Feb 12 2004
Thank you for your patience. I am working with the originator of ab7747 to help determine what may be going on with this particular antibody. I would like to confirm, with this batch of ab7747 you never saw an Il-8 band and the only band you saw was at approximately 40 kDa, correct? Did you ever try other positive cells as activated macrophages? Can you give me the lot number that you received - it will be on the vial. Many thanks.
Answered on Feb 19 2004