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LOT NUMBER GR21753-2 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM Non-specific band WB from IP samples show no band for 37kDa zone also, the input lane shows two bands (strong one at 50kDa and a weak band at 37kDa) SAMPLE Rat primary hippocampal cell culture PRIMARY ANTIBODY 1:1000 diluted in 0,5% milk TBS-T Washed 3x 10min with TBS-T DETECTION METHOD ECF, 5min max ANTIBODY STORAGE CONDITIONS 4ºC (short-term) -20ºC (aliquots) SAMPLE PREPARATION RIPA supplemented with proteases inhibitors (CLAP and PMSF), phoshpatase inhibitor (NaF), DTT and RNase inhibitor Samples from IP were dissolved with sample buffer 2x and heated at 95ºC for 5min AMOUNT OF PROTEIN LOADED 50ug for Input 1000ug for IP ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE, tris-bicine, 12,5% polyacrylamide TRANSFER AND BLOCKING CONDITIONS CAPS-methanol, 40V o.n. 4ºC blocked with 5% milk TBS-Tween SECONDARY ANTIBODY Anti-Mouse 1:20000 diluted in 0,5% milk TBS-T Washed 3x 10min with TBS-T HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? Adding the flow-through samples to the WB
Asked on Oct 28 2011
Thank you for your enquiry regarding ab6102 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody. Band at 37 kDa in input sample indicate that the antibody is working fine and further optimizations in antibody dilutions could help getting clean band at right size. Based on the information provided I presume that antibody is unable to IP the protein. So in order to understand the problem I would like to ask few questions; - What type of beads were used for IP? e.g. protein A or G - How the IP was performed? Have you mixed antibody beads and lysates once, or beads and antibody first then lysates or antibody lysates and then beads? - For how long the mixture was incubated? - Can we define the problem as no bands in IP or multiple bands; because loading 1000 ug protein per well in WB is way too high. If it is not true then I am presuming that this is the amount of protein (lysates) you have used to mix with antibody and beads. - 1/1000 dilution means the antibody was used at 1ug/ml which is very low; I would recommend using ab at 5 ug/ml and 10ug/ml. - Could you send us an image? I hope the suggestion of conc. will help to improve the results. I will also look forward receiving reply to my questions soon.
Answered on Oct 28 2011