Anti-HDAC4 antibody - ChIP Grade (ab1437)

Could you please tell the region that this antibody binds to?

ANSWER:

Thank you for calling Abcam earlier today. I have recieved a reply from the lab about the region where ab1437 binds. They have told me that the anitbody was generated using a synthetic peptide corresponding to residues surrounding amino acid 10 of human HDAC-4  If there is anything I can help you with, please let me know.  

Thank you for your answer. Yes I have detected the expression of beta-actin (with another Abcam mouse monoclonal antibody) and the expression was very good. I hope this helps. I look forward to hearing from you again soon.

ANSWER:

Thank you for getting back to me and clarifying this issue. Once again I am surprised by your observed lack of expression of HDAC4 in HeLa cells given the literature that reports its expression in these cells.

I would like to offer you a replacement vial or credit note provided that the antibody was purchased within the past 90 days. If this is the case please e-mail me details of the date of purchase and order order number and I will make the necessary arrangements.

I look forward to hearing from you.

BATCH NUMBER 163103 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM No band

SAMPLE Human cell extract from HeLa cells (total lysate)

PRIMARY ANTIBODY ABCAM AB 1437-100 antibody at 1 mcg/ml in TTBS1x + Milk 5%. Incubation at room temperature for 4hours Washings in TTBS 1X: 2 rapid washings, followed by 1 washing RT for 15 minutes, followed by 2 washings of 5 minutes/each at room temperature.

DETECTION METHOD ECL (Amersham).

POSITIVE AND NEGATIVE CONTROLS USED Positive control: HeLa cells whole lysate

ANTIBODY STORAGE CONDITIONS Stored at -20C upon arrival

SAMPLE PREPARATION Cell-Signaling cell lysis buffer with the addition of PMSF. Sample heated at 100C for 8 minutes before loading.

AMOUNT OF PROTEIN LOADED 50 micrograms

ELECTROPHORESIS/GEL CONDITIONS PAGEr Gold Precast Gels 12% Tris-Glycine gels Cambrex (Cat# 59503)

TRANSFER AND BLOCKING CONDITIONS Transfer with Tris-Glycine Buffer 1X + Methanol 20%. Blocking with TTBS 1x + 5% milk blocking ON +4C

SECONDARY ANTIBODY [another company] anti-rabbit, horseradish peroxidase linked whole antibody (from donkey), cat. #NA934V. 1:2500 in TTBS 1x + 5% milk. Incubation at RT for 2 hrs. Washings as above.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. I have read your technical questionaire and you are largely following a protocol that I would recommend. Furthermore according to the literature HeLa cells express HDAC4. However, I would appreciate it if you could tell me whether you have been able to detect other cytosolic proteins using this method of extraction. I am not aware of the constituents of the cell signalling lysis buffer. I would recommend that you use a RIPA buffer extraction and appreciate your comments on this.

I look forward to hearing from you.

Customer is using this antibody in Western blotting with mouse muscle lysate and is detected a single band at approx. 100 kDa. Has this been seen before with this antibody (it states that this antibody detects ~140 kDa histone deacetylase 4)?

ANSWER:

Thank you for your enquiry. The originator of this antibody has told me that they sometimes see two weaker bands at 80-100 kDa and another one at 40-60 kDa. They think these could be the cleavage fragments of HDAC4.

Please note that some tissues may not express HDAC-4 and 5 protein or it is expressed at extremely low amounts. HDAC-1,2,3,6,7 are usually much more abundant (could be ~100X more) than HDAC-4 & 5. We would strongly suggest using positive control (HeLa lysate).

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.