Anti-KAT13B / AIB1 antibody - ChIP Grade (ab2831)

BATCH NUMBER 169016 ORDER NUMBER 134911

DESCRIPTION OF THE PROBLEM No signal or weak signal Only after 1 hour exposition I see a weak band with 80 ?g of a T47D lysate and a little bit stronger band with a 293T extract (transfected cells with human NCoA3). There are some strong unspecific bands at 100 kDa and higher and also at 75 kDa and below

SAMPLE I used 293T and T47 cell extracts

PRIMARY ANTIBODY Rabbit polycolnal to AIB1 (ab2831) in blocking solution, incuabtion overnight at 4?C dilution 1:500 and 1:000 washing: 3x with TBST

DETECTION METHOD ECLplus (Amersham): incubation 5 min

POSITIVE AND NEGATIVE CONTROLS USED positive control should be a T47D cell lysate (as described in your data sheet

ANTIBODY STORAGE CONDITIONS Aliquots stored at -20?C

SAMPLE PREPARATION Buffer: 20 mM Tris/HCl, pH 8; 100 mM NaCl, 1 mM EDTA, 1 mM DTT, 0,2% (v/v) NP-40, 10% Glycerol Protease Inhibitor: Complete EDTA-free (Roche) Phosphataseinhibitors: Phosphatase Inhibitor Cocktail 1 and 2 (Sigma) Denaturation: 5 min 95?C

AMOUNT OF PROTEIN LOADED I used 80 ?g of 293T cell lysate and 20, 40 and 80 ?g of T47D cell lysate

ELECTROPHORESIS/GEL CONDITIONS reducing conditions, 8% SDS gel. run with 80-120 V

TRANSFER AND BLOCKING CONDITIONS transfer with a 3 buffer system, conditons: 1 h, 100 mA (PVDF membrane 9x7 cm) blocking agent: 5% milk powder in TBST (1h, room temparature)

SECONDARY ANTIBODY anti rabbit HRP-linked (another company) from donkey, dilution: 1:10000 in blocking solution, incubation: 1h, room temperature washing: 3x with TBST

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? I change the dilution from 1:1000 to 1:500

ADDITIONAL NOTES I used the same antibody also in a ChIP experiment and I get also no good results, but in this case it could also be a optimization problem of my ChIP procedure

ANSWER:

Thank you for the protocol details that you have provided and I'm sorry to hear that you are experiencing difficulty with ab2831. The antibody was characterized for application in Western blotting using T47D cell lysate and a band at 155 kDa was detected. You certainly are loading a sufficient amount of lysate and a suggestion would be to try using nuclear extract instead of whole cell lysate, but the antibody's originator was able to detect AIB1 in the T47D cell lysate. Just to check - is your secondary antibody working properly with other primaries?

There have not been any problems with the batch that you received and it sounds to me as if there may be a problem with the particular that you received. I would be happy to send you a free of charge replacement vial or can issue you a credit note or refund. Please let me know how you would like to proceed and I look forward to hearing from you.