Question (14232) | Anti-RASSF1a antibody [3F3] (ab23950)

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Question

BATCH NUMBER N/A ORDER NUMBER 102540 DESCRIPTION OF THE PROBLEM Non-specific band appears at 39kDa. SAMPLE I have used cell extracts from cell lines with known lack of RASSF1A expression (NCI-H1299, A549) as well as positives (NCI-H1792 and HeLa) in a western blotting expt. A band corresponding to 39 kDA appeared in every lane. I probed a western with extracts from HEK293 cells transfected with HA-tagged RASSF1A and RASSF1C. The antibody did not pickup the expression from those constructs, although the HA did. I also probed lysates from RASSF1A siRNA transfected HeLa cells. No joy! PRIMARY ANTIBODY 1:500 and 1:1000, 60 min, 3x5 min PBS-tween washes DETECTION METHOD ECLPlus POSITIVE AND NEGATIVE CONTROLS USED Cell lines with known RASSF1A expression status ANTIBODY STORAGE CONDITIONS As recommended 4degrees SAMPLE PREPARATION 1% Triton-X in Tris buffer. Roche complete protease inhibitors were used in the lysate buffer. SDS loading buffer added to upto 10 micrograms of lysates and boiled before loading on a 12% SDS-PAGE AMOUNT OF PROTEIN LOADED 10 micrograms ELECTROPHORESIS/GEL CONDITIONS 12% SDS-PAGE TRANSFER AND BLOCKING CONDITIONS 1 hr Tris-glycine-methanol. Blocked in 5% milk-PBS-Tween SECONDARY ANTIBODY [a competitor] rabbit antimouse 1:5000, 30 min, 3x5 min PBS-tween washes 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? No ADDITIONAL NOTES will send images when required

Answer

I have just received feedback from the source of ab23950 and have been informed that we are studying ab23950 with two university groups. Those groups used cell lysate of HeLa as positive control and 293cells transfected with HA-tagged RASSF 1A and RASSF 1C gene. In both groups, there was no problem in detecting a specific band in transfected cells. We are still in the process of re-confirming that the current batch works with HeLa cell lysate as positive control to make sure that the antibody is specific for exogeneous RASSF1a and for endogenous RASSF1a. We have of course previous QC data of this antibody where it detected a band with HeLa and tissues from mouse with no non-specific bands. I apologise for the delay in re-testing this antibody and can offer you a credit note or refund or replacement if you purchased the antibody in the last 90days,

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