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Question (12488) | Anti-Insulin Receptor (phospho Y972) antibody (ab5678)

BATCH NUMBER 99451 ORDER NUMBER V06053 DESCRIPTION OF THE PROBLEM Non-specific band, wrong band size. SAMPLE Cell extract. PRIMARY ANTIBODY Primary antibody is used here ab5678 and at the dilution at 0.5ug/ml in 5% TBS milk for 2hrs. at RT. DETECTION METHOD ECL for 2mins. POSITIVE AND NEGATIVE CONTROLS USED No positive and negative control is used here. ANTIBODY STORAGE CONDITIONS After opening the pack, the antibody is stored at -20C SAMPLE PREPARATION In the lysis buffer, protease inhibitors are already added.And once the sample is prepared, after adding 4X sample buffer,it is to be heated upto 100C for 5 mins. AMOUNT OF PROTEIN LOADED In microgram amount but exactly the OD in spectrophotometer at 595nm, is arround 0.4. ELECTROPHORESIS/GEL CONDITIONS Reducing gel and 10% sds gel. TRANSFER AND BLOCKING CONDITIONS 1.Transfer buffer is used as per the protocol and over night transfer on nitrocellulose membrane. 2. 5%TBS milk is used for blocking the membrane after the transfer for one hour at room temperature(RT). SECONDARY ANTIBODY Secondary is used Goat-Anti-Rabbit (1:1000) from [a competitor] with 5%TBS milk for one hour at RT. 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 WHAT STEPS HAVE YOU ALTERED? No steps. ADDITIONAL NOTES Nothing at this point but want to know the your at the earliest and all the possible points to get my result back.

I'm sorry to hear you are having a problem with ab5678. It would be helpful if you could also tell me the type of cells you are using, what size bands you see, and you could also attach a picture of your blot and that would be very helpful. Using the information you have provided to me so far, I would like to suggest the following modifications to your protocol: 1) a positive and negative control would be very helpful such as cells transfected with human insulin receptor +/- insulin treatment, 2) try varying the concentration of antibody and using it at 4 degrees overnight, 3) remove the milk from the primary and secondary, and then if there are too many bands add milk to 0.5 - 1%, 4) figure out the amount of protein you are adding and load 20 to 30 ug protein per lane, 5) try using the secondary at 1:10,000. Please let me know if this helps and do not hesitate to contact us for further advice,