Anti-KDM4A / JHDM3A / JMJD2A antibody - ChIP Grade (ab24545)

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 222875

DESCRIPTION OF THE PROBLEM more non-specific bands, and weak target band

SAMPLE human/Hek293 cell/cell extract human/A549 cell/nuclear extract mouse/HIF+ MEF cell/cell extract

PRIMARY ANTIBODY Abcam/rabbit/diluted in blocking buffer/1:2000 dilution/incubate overnight

Washing: 5min TBS-Twin/ 3 times

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED transfected pSG5-Flag-JMJD2A (JMJD2A overexpressed sample) (can be strongly detected by anti-Flag antibody)

ANTIBODY STORAGE CONDITIONS aliquot and store at -20 degree C

SAMPLE PREPARATION RIPA buffer with cocktail protease inhibitor/boiled 5min with SDS loading buffer before loaded to the gel

AMOUNT OF PROTEIN LOADED 50 microgram

ELECTROPHORESIS/GEL CONDITIONS 15% and 5% Tris-HCl SDS gel

TRANSFER AND BLOCKING CONDITIONS For each 1L transfer buffer: 100ml BioRad 10*TG (Tris/Glycine buffer), 200ml methanol, 700ml miliQ water Transfer time: 30v overnight in cold room (4 degree C) Blocking agent: 5% milk in TBS buffer

SECONDARY ANTIBODY Santa Cruz Biotechnology/anti-rabbit/diluted in blocking buffer/1:5000 dilution/incubate 1 hour at room temperature Wash: 10min TBS-T/3 times, 10min TBS once, 5min water once

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? different cell lines

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been experiencing problems with ab24545 in western blot.

Often it is possible to make suggestions that help resolve problems. We will happily offer technical support and in the event that a product is not functioning in the applications cited on the product data sheet (and the problem has been reported within 120 days of purchase), and if it appears that the antibody is at fault, a free replacement/credit note/refund will be offered.

I can confirm that this product will work well in human samples. However, it has yet to be tested in mouse samples and we can not guarantee you will obtain good results. I have looked through the protocols you used and have a few questions and suggestions that might help you resolve the problem.

- One of the most common cause of non-specific bands is there is an over loading of protein. We recommend using 20-40ug per well. This will eliminate the non-specific bands. One other reason is that the protein target may form multimers. Try boiling in SDS-Page for 10 minutes rather than 5 minutes to disrupt multimers.

- The target in your protein sample might have been digested (more likely if the bands are of lower molecular weight). Please make sure that you incorporate sufficient and fresh protease inhibitors in your sample buffer.

- As this antibody is supposed to detect a band of approximately 120-130kDa, using an 8% gel is recommended so that the proper molecular weight proteins can be separated accordingly. - At Abcam, almost all of our products has been tested with 5% BSA. Sometimes, a different blocking buffer might cause cross reaction between the blocking agent and antibodies, therefore causing weak bands or no bands at all. I would recommend trying 5% BSA, if you have not already done so.

- Can you confirm that the second antibody you used also managed to produce good results with other primary antibodies? Could it be causing the weak signal? Are the non-specific bands weak or strong? If the non-specific bands are stronger, then the protein of interest is not abundantly present in the cell extract.

- How much Tween was in the TBST solution? In general, a 0.05% TBST would be sufficient as a washing buffer. Also, was the washing done 15min x 3 times? This is because over washing will eventually cause you to lose signals and insufficient washes will not be able to rid the non-specific bands.

I am sorry for the string of questions but I hope the above recommendations may already help you. If you have already tried the above suggestions and still experience problems, please do not hesitate to contact me with the answers to the above (including an image), and details of your order (contact information, shipping address/purchasing agent, etc.). Also, please advice me on how you would like to proceed with your enquiry, so that I can immediately arrange for a replacement or refund for you.