Anti-CD9 antibody [MEM-61] (ab2215)

BATCH NUMBER 163262 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM lots of bands and not at the right predicted molecular weight for cd9

SAMPLE as a positive control used full lysed DCs (NP40 lysis buffer, protease inhibitors: the samples however are the different fractions of a cell separation using a continuous iodixanol density gradient solution: trying to find a DC-SIGN/CD9 positive endosomal compartment. (used human DCs)

PRIMARY ANTIBODY cd9 (Abcam) have tried it at different dilutions: 1/100, 1/500, 1/1000 dilutant: PBS/0.1% tween incubation time: 1 hour wash 3X for 10 min with PBS/tween

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED positive: full DCs

ANTIBODY STORAGE CONDITIONS -20 C

SAMPLE PREPARATION 15ul of sample (either fractions or full cell lysis) and 15ul of 2X loading buffer (no DTT) (and samples not heated)

AMOUNT OF PROTEIN LOADED not exactly known, but in the full cell lysis there should be a lot of it

ELECTROPHORESIS/GEL CONDITIONS non-reducing gel, 4-12%

TRANSFER AND BLOCKING CONDITIONS transfer onto nitrocellulose, 100mA in transfer buffer blocking agent: milk powder

SECONDARY ANTIBODY anti-mouse HRP (Sigma) in PBS/0.1% Tween (2ul/5ml) 1 hour wash 3X for 10 min with PBS/Tween

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 6 HAVE YOU RUN A "NO PRIMARY" CONTROL? Yes DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? concentration of anitbody and of loaded protein (have loaded as much as possible), incubation times (have left it for less and longer than an hour)

ADDITIONAL NOTES I have seen that you have another CD 9 antibody which is a rabbit anti human Ab, and I would like to perhaps try that one instead. I am doing a 3-month MSc project and thus have very limited time, which I cannot afford anymore to waste on trying to get an antibody to work. Thanks in advance.

ANSWER:

I'm very sorry to hear you are experiencing a problem with ab2215. I appreciate the urgency in resolving this problem due to the short time you gave in the laboratory, and I believe that with the following protocol tips you will have improved results with ab2215.

Often non specific bands are due to the following problems:

1) protein degradation. we recommend the following inihibitors to be present in the lysis buffer: Aprotinin 2 µg/ml , Leupeptin 5-10 µg/ml, Antipain 2-10 µg/ml, Pepstatin A 1 µg/ml, Na-Fluoride 5-10 mM, phosphatase Orthovanadate 1 mM , PMSF 1 mM. If one of those has degraded in your samples this may explain the problem. Furthermore as you are performing a complicated fraction protocol this provides further opportunities for the proteases to act if they are not totally inhibited (please make sure all steps are carried out at 4C too).

2) the antibody is not binding to the membrane adequately. This can be due to too little or too much blocking (what percentage do you use and how long is your incubation for?). This can make a hughe difference in the binding ability of the antibody. I would recommend to try 5%BSA for 1 hour and incubating the membrane in TBST only overnight (TBST is better than PBST). The problem can further be due to too much primary antibody, so it is best to dilute the antibody more and incubate longer, to provide a slow but targeted binding to the protein of interrest (you may also find you can dilute the secondary more too).

I hope the above recommendations will help you. Please do not hesitate to contact me again if I can be of further help,