Anti-HDAC6 antibody (ab1440)

I need more detailed information about the immunohistochemical procedure concerning yuor product Anti-HDAC6 ab1440 . I'm going to use ab1440 on formalin-fixed, paraffin-embedded rat tissues and I want to know the exact antigen retrival procedure requested (if necessary). Could you also suggest me a particular rat tissue that constitutively shows prominent HDAC6-IHC expression (positive control)? Thank you for your precious information Best Regards

ANSWER:

Thank you for your enquiry.

I have been in touch with our lab team and they have informed me that for the application of this antibody by IHC using paraffin embedded sections antigen retrieval should be performed by heating sections in a microwave for 1 minute at high power before 10 minutes at medium power in 10 mM sodium citrate buffer. Keep slides fully immersed.

For a positive control; human tonsil tissue sections can be used.

I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

BATCH NUMBER 163987 ORDER NUMBER 122931

DESCRIPTION OF THE PROBLEM The size of HDAC 6 should be around 134 kDA, but instead, I get 80 kDA each time

SAMPLE mouse,whole cell extract

PRIMARY ANTIBODY 1:1000

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED we re-blot the same membrane with [competitor's]HDAc6 Ab and got correct size band

ANTIBODY STORAGE CONDITIONS -20

SAMPLE PREPARATION standard buffer with protease inhibitors

AMOUNT OF PROTEIN LOADED 100ug

ELECTROPHORESIS/GEL CONDITIONS 6% reducing gel

TRANSFER AND BLOCKING CONDITIONS standard

SECONDARY ANTIBODY standard protocol

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? Yes

WHAT STEPS HAVE YOU ALTERED? no

ANSWER:

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with ab1440. This antibody has been tested with HeLa cell lysate (20 ug) and a ~134 kDa band was detected. The antibody should with mouse samples as well. Can you tell me specifically the type of mouse samples that you are using? Can you also send an image of your blot?

Thank you and I look forward to hearing from you.

Could you tell me the immunogen for this antibody? I check the other 2 Anti-HDAC6 antibody you have. They all provide the epitope used to generated the antibody, but this one not.

ANSWER:

The immunogen used was a synthetic peptide mapping to Mouse HDAC-6 N-terminus.

If you have any additional questions, please contact us again.

I have had the exact same problem with HDAC antibodies 4 and 6. I have failed to detect them on a western using Hela extract. The blots were completely blank. I went upto 20ug of Hela extract. As a control, I blotted with HDAC 1 antibody. HDAC 1 gives a nice band of the right size; HDAC4 and 6 lanes are blank. Experiment was repeated 2 times. Got the same result.

ANSWER:

Thank you for the information. I have instructed our accounting department to issue you a refund for ab1440 and ab1437. This process does take a little while and if you have any questions regarding the refund, please contact our accounts department at accounts@abcam.com. If you have any additional questions, please let me know.

Sorry for the delayed response. I was out of town. Here is the information you requested. 1) The blots were completely blank. I went upto 20ug of Hela extract.(your antibodies to HDAC1,3 worked great for me. I have given positive reviews for them on your website) 2) Dilutions: 0.5ug-2ug/ml 3) Lot number: 80822

I had also purchased Ab1437 in the same order. I submitted a request for a refund, but have not heard from Tech services on that one. The lot number for that is 75530

ANSWER:

Thank you for the information regarding ab1440. I searched through our system and I don't have a record of an enquiry from you regarding ab1437. Can you please send me a description of the problem with ab1437 and your protocol? I will then investigate both ab1440 and ab1437 together. Thank you, and I look forward to hearing from you.