Overview

  • Product name
  • Description
    Rabbit polyclonal to HDAC4
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Human
  • Immunogen

    Synthetic peptide corresponding to Human HDAC4 aa 1-18.
    Sequence:

    MSSQSHPDGLSGRDQPVE

  • Positive control
    • Hela cell lysate. This antibody gave a positive result when used in the following methanol fixed cell lines: HepG2

Properties

Applications

Our Abpromise guarantee covers the use of ab16339 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IHC-P 1/20.
ICC/IF Use a concentration of 5 µg/ml.
WB 1/1000. Predicted molecular weight: 140 kDa.

Target

  • Function
    Responsible for the deacetylation of lysine residues on the N-terminal part of the core histones (H2A, H2B, H3 and H4). Histone deacetylation gives a tag for epigenetic repression and plays an important role in transcriptional regulation, cell cycle progression and developmental events. Histone deacetylases act via the formation of large multiprotein complexes. Involved in muscle maturation via its interaction with the myocyte enhancer factors such as MEF2A, MEF2C and MEF2D.
  • Tissue specificity
    Ubiquitous.
  • Involvement in disease
    Defects in HDAC4 are the cause of brachydactyly-mental retardation syndrome (BDMR) [MIM:600430]. A syndrome resembling the physical anomalies found in Albright hereditary osteodystrophy. Common features are mild facial dysmorphism, congenital heart defects, distinct brachydactyly type E, mental retardation, developmental delay, seizures, autism spectrum disorder, and stocky build. Soft tissue ossification is absent, and there are no abnormalities in parathyroid hormone or calcium metabolism.
  • Sequence similarities
    Belongs to the histone deacetylase family. HD type 2 subfamily.
  • Domain
    The nuclear export sequence mediates the shuttling between the nucleus and the cytoplasm.
  • Post-translational
    modifications
    Phosphorylated by CaMK4 at Ser-246, Ser-467 and Ser-632. Phosphorylation at other residues is required for the interaction with 14-3-3.
    Sumoylation on Lys-559 is promoted by the E3 SUMO-protein ligase RANBP2, and prevented by phosphorylation by CaMK4.
  • Cellular localization
    Nucleus. Cytoplasm. Shuttles between the nucleus and the cytoplasm. Upon muscle cells differentiation, it accumulates in the nuclei of myotubes, suggesting a positive role of nuclear HDAC4 in muscle differentiation. The export to cytoplasm depends on the interaction with a 14-3-3 chaperone protein and is due to its phosphorylation at Ser-246, Ser-467 and Ser-632 by CaMK4. The nuclear localization probably depends on sumoylation.
  • Information by UniProt
  • Database links
  • Alternative names
    • AHO3 antibody
    • BDMR antibody
    • EC 3.5.1.98 antibody
    • HA6116 antibody
    • HD 4 antibody
    • HD4 antibody
    • HDAC 4 antibody
    • HDAC A antibody
    • HDAC4 antibody
    • HDAC4_HUMAN antibody
    • HDACA antibody
    • Histone deacetylase 4 antibody
    • Histone Deacetylase A antibody
    • KIAA0288 antibody
    see all

Images

  • ab16339 at 1/1000 detecting HDAC4 from HeLa cell lysate by Western blot
  • Immunohistochemistry was performed on normal biopsies of deparaffinized Human skin tissue. To expose target proteins, heat induced antigen retrieval was performed using 10mM sodium citrate (pH6.0) buffer, microwaved for 8-15 minutes. Following antigen retrieval tissues were blocked in 3% BSA-PBS for 30 minutes at room temperature. Tissues were then probed at a dilution of 1:20 with a Rabbit Polyclonal Antibody recognizing HDAC4 (ab16339 ) or without primary antibody (negative control) overnight at 4°C in a humidified chamber. Tissues were washed extensively with PBST and endogenous peroxidase activity was quenched with a peroxidase suppressor. Detection was performed using a biotin-conjugated secondary antibody and SA-HRP, followed by colorimetric detection using DAB. Tissues were counterstained with hematoxylin and prepped for mounting.

  • ab16339 stained HepG2 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab16339 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

ab16339 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Answer

Thank you for contacting us.

I wish to apologize for the confusion as I had made this mistake when creating the recall email. The correct immunogen does correspond to aa1-18 of human HDAC4 and not to the peptide stated above. Thank you very much for alerting me to this mistake.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you very much for sharing the results you obtained with ab32073. I am very pleased to hear that it has worked well for you.

It would be very useful for other customers if you could share this information in the form of an Abreview? This should only take 5-10 minutes to complete and will allow other customers to judge if the antibody is appropriate for their experiments. As a reward you will earn Abpoints which can be converted to Amazon vouchers, or credit off future orders with Abcam.

More information on the Abreview system and Abpoints can be found from the following link:

https://www.abcam.com/abreviews

https://www.abcam.com/abpoints

If I can be of any further assistance, please do let me know.

Until then, I wish you all the best with your research.

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Answer

Thank you for your reply and understanding. I emailed as quickly as I could after receiving the information so that you wouldn't be running blots unnecessarily.

I have now arranged for the rabbit monoclonal ab32073 to be sent to you. This is on the order number 1179028 (Purchase order number FOCR 4507028970/50A PRU2910995-1). This should reach you tomorrow.



Please do let me know how the ab32073 performs in your experiments.

In the meantime, if I can be of any further assistance, please do not hesitate to let me know.

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Answer

Thank you for getting back to me.

As I think I mentioned to you, I was consulting with the source of this antibody to get confirmation of the immunogen used to raise the antibody.

Unfortunately, they have gone back in their records and found that an error was made. As your results have supported, the antibody was actually raised against the N-terminal portion of the HDAC4 protein. I am very sorry for this error and the inconvenience it has caused you. We will be updating our datasheet with this information, as well as emailing all former customers of this updated information. Thank you for bringing this to our attention.

I will of course be able to send you the rabbit monoclonal ab32073 if you would like to try it. I am sorry but when I suggested it I was under the impression that you were using human sequences for your construct. You are correct that we have found this antibody not to be able to detect the mouse protein. This is as you have mentioned, with the endogenously expressed protein and with the over expressed constructs you are using this may be different. The immunogen used to raise the antibody has two alterations between the human and mouse sequence out of a total of 17 residues. We would therefore normally predict that there would be cross reactivity however I cannot guarantee this. If you find this antibody not to work in your experiments I would of course be able to provide you with a refund or credit note.

If you would like me to send ab32073 please do let me know. If you let me know before 3 pm today it can be with you tomorrow.

Alternatively, I can offer a credit note or refund.

I am very sorry for the inconvenience this error has caused you. look forward to hearing how you would like to proceed.

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Answer

Thank you for getting back to me. If you wouldn't mind testing ab16339 again I think that would be worthwhile, just to confirm there was no mix up along the way. If you get the same results again I'd be happy to send ab32073 if you would like to try it.

I was just wondering, there is no change when aliquotting ab16339 to be frozen that any of the aliquots got mixed up with the other antibody?

I'd be interested to see the results of both ab16339 and the xxxxx antibody. Hopefully the results are as would be expected this time.

I look forward to receiving your reply.

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Question

Dear xxxxx,

Further to our telephone conversation, please find a powerpoint presentation for HDAC4 consisting of three slides.

Slide1, shows the constructs I have generated ranging from the full-length HDAC4 protein (H4-1) and various N-terminal (H4-2, H4-6 to H4-11) or C-terminal (H4-3 to H4-5) deletion mutants.

Slide 2, shows them in vitro translated using a rabbit reticulolysate system.

Slide 3, shows a HEK293T transfection with these constructs in induced or un-induced cells which over-express mutant Huntingtin (i.e. – or +), the disease in which we work on. As you will see I have probed blots with either the Sigma antibody DM-15 (top panels) or your abcam antibody ab16339 (bottom panels), and basically both gel sets look pretty much identical which suggests to me that the ab16339 clone you sell has to be N-terminal too. Therefore, as you will see there is no signal in mock untransfected cells (as expected) with either antibody. Both antibodies (as expected) recognise cells transfected with the full-length protein (H4-1). Then Sigxxxx(aa14-28) as expected should only recognise N-terminal fragments, i.e. H4-3, H4-4, and H4-5, whereas ab16339 (aa1071-1084) should only recognise H4-2, H4-6, H4-8, H4-9, H4-10 and H4-11.

This, however, is not the case. In fact ab16339 recognises exactly the same as xxx implying that its epitope must indeed by N-terminal. As explained, there is no chance I have put the wrong antibodies on the wrong blots as the background (non-specific) banding profile is different between the xxx and ab16339 probed blots.

Please let me know if you require any further information regarding this matter.

With best regards,

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Answer

Thank you for providing that information. It has made understanding the problems encountered in your experiment much easier to understand.

As we discussed over the phone, it is very unlikely that you have been provided with the wrong vial of antibody. From the results you have shared with me, I think the two most likely scenarios is that either the incorrect antibody was loaded on the membrane or that the antibody immunogen information is incorrect. I am currently looking into the latter.

Although you have said that you are sure that the correct antibody was loaded the non-specific staining between the two antibodies does look very similar, with just a difference in intensity of the bands. This does suggest that it may be the same antibody. Can I assume both are raised in rabbit and the secondary antibody would therefore not be able to distinguish between the N-terminal antibody and ab16339?

You mention another antibody, SC-H4, how did this perform in your experiment?

In order to try to ascertain which reason is likely to be responsible for these unexpected results I can provide you with a different antibody to try free of charge. The rabbit monoclonal https://www.abcam.com/HDAC4-antibody-Y417-ab32073.html which is also raised against the C-terminal domain of the HDAC4.

If you would like me to arrange this for you please do let me know. I currently have the order number xxxx (Purchase order number xxxxx) of the 5th of September 2012, is this correct?

I look forward to receiving your reply.

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