Anti-HDAC4 (phospho S632) antibody (ab39408)

There are two bands (120 kd and 140 kd) in western blot using HDAC5 antibody( from another Vendor). It is likely that 140 kd is the p-HDAC5 and 120 kD is the HDAC5 (non-phosphorylation). Reference also reported that p-HDAC has bands shift. Do you notice any band shift for p-HDAC using your HDAC antibodies?

ANSWER:

Thank you for your reply. It is definitely possible that you may notice a shift in the molecular weight. We have not tested this extensively with our general HDAC5 antibodies, but it could explain the extra bands in lane three of the WB image for ab11969. I hope this helps, please let me know if you have any additional questions or concerns.

Sorry I made a mistake about the species. The L6 cells are from rat instead of Rabbit. I use the 5% milk as blocking solution but I use 5% BSA to dilute the primary Ab as some protocol suggested.
A nuclear receptor gene was transduced in L6 cells. I cannot tell if the Western blot results are similar in the control cells( you can compare the two lanes in the low blot, or you can compare odd lane with even lane).
Thanks.

ANSWER:

Thank you very much for your reply.
It looks like there may be a band around the expected molecular weight that is more visible in the even lanes compared to the odd lanes (except for lane 9, it looks like the band is also in this lane). However it is quite difficult to know for sure due to the other bands on the blot. I would suggest trying some of the alterations mentioned before (the BSA blocking, 1:1000 dilution, and nitrocellulose if available).
I also understand if you would prefer to try a new vial of antibody, however we only have the same lot in stock that you have been working with. Please let me know if you would like to try a replacement antibody, or alternatively I can issue a credit or refund.
I look forward to hearing from you. Let me know if you have any questions or if there is anything else that we can do for you.

Please see the following to your inquiry:
Thanks.


1) What kind of samples were used? What species and cell/tissue type? Were they treated to induce phosphorylation or untreated?

Cultured L6 cells(rabbit) lysis. The cells are stably expressed another gene and the control cells (vector control).

2) How much protein was loaded per lane?

About 20-40 ug
3) What kind of blocking solution was used (e.g. milk, BSA, etc)?

5% milk.

4) What dilution/concentration of antibody was used? How long was the antibody incubated with the membrane?

1:500 dilution. 4C O/N.

5) What type of membrane was used?

Millipore PVDF.

6) What solution do you use to wash the membrane?

TBS with 0.1% Tween 20.

There are three known human isoforms of this protein, which span from 112-122 kDa expected molecular weight. Do you know if your samples could contain all of these isoforms?

http://www.uniprot.org/uniprot/Q9UQL6

ANSWER:

Thank you very much for your reply and for sending this additional information.
I do have a couple more questions. What gene is being transduced in these L6 cells? Were the Western blot results similar in the control cells?
We have not yet tested this antibody in rabbit samples, so we can not guarantee that the results will be optimal, but I do have a few suggestions that I hope will improve the results.
I would recommend using 5% BSA for blocking, as milk can be problematic with some phospho-specific antibodies. Milk contains phospho-proteins and phosphatases, both of which can contribute to background or reduce specific signal in Western blot. You can also further dilute the antibody to 1:1000 to reduce non-specific binding. If you have nitrocellulose membranes available, I would also recommend using these as they often give less background than PVDF.
I cannot find much information about the HDAC5 isoforms in rabbit, but it is possible that some of these bands represent different isoforms, and they may still be visible even after making the alterations above.
I hope these suggestions will be useful, and please keep me updated about any new results. Please let me know if you have any questions or if there is anything else that we can do for you.

Dear Sir:
I use the Anti-HDAC5 (phospho S259) antibody (ab53693) for Western blot. There are many bands in Western blot film ( see attachment, two time of Western blot) instead of one band as in you website. It is difficult to identify which band is the specific band. I am not sure if the problem is relative to my method or to the quality of the Abs. I get one band for Anti-HDAC4 (phospho S632) antibody (ab39408) using the same method. Please give me some suggestions. Thanks.

ANSWER:

Thank you for contacting us and letting us know about the trouble with ab53693.
I do have a few questions so that I can better understand the situation:
1) What kind of samples were used? What species and cell/tissue type? Were they treated to induce phosphorylation or untreated?
2) How much protein was loaded per lane?
3) What kind of blocking solution was used (e.g. milk, BSA, etc)?
4) What dilution/concentration of antibody was used? How long was the antibody incubated with the membrane?
5) What type of membrane was used?
6) What solution do you use to wash the membrane?
There are three known human isoforms of this protein, which span from 112-122 kDa expected molecular weight. Do you know if your samples could contain all of these isoforms?
http://www.uniprot.org/uniprot/Q9UQL6
I look forward to hearing from you and resolving this promptly. We do guarantee this antibody to work in Western blot with rat and human samples, so I would be happy to send a replacement antibody if necessary.
Please let me know if you have any questions or if there is anything else that we can do for you.

Dear technical team, Our customer would like to purchase this antibody and test ICC/IF. If you have information when this item released, please let me know. The custmoer wants to receive discount code and purchase this item. Check this please. Best regards,

ANSWER:

Thank you for contacting us. We have only tested this antibody in HeLa cells for IF. The results were not very convincing, but if your customer is still interested in testing it with a different cell line, I can offer him/her a testing discount to perform an ICC/IF assay. For that, please send me the customer details (name, email and institution). I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.