Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab4729)

I recently ordered two vials of the H3K27ac antibody (cat #: ab4729, Lot #: GR71158-2). As we do with all histone modification antibodies in our lab, I used Active Motif's MODified Histone peptide array to test the specificity of the antibody before using it for a ChIP experiment. I've attached a powerpoint file with the associated data for this antibody as well as another antibody that I tested at the same time. For each antibody, there are two slides. I used Active Motif's free peptide array analysis software to do the data analysis. The first slide shows the graphical data of what modifications were hits on the array. The program presents the top 10 results. The second slide is an image of the peptide array with blue circles highlighting where the modifications that the antibody is supposed to specific for, were present on the array. As you can tell, the antibody lot for H3K27ac was not specific at all for H3K27ac.  As reference I also have attached the data from the Millipore H3K4me3 antibody that I tested. 

I have found in the literature that your antibody is commonly used for ChIP-sequencing experiments and I was hoping to do the same. 

Would you be able to send me other (different) lots to test specificity?  Let me know how we can proceed since we've ordered two vials of this antibody and it is not usable. If not, is there a way that our lab could be refunded?

ANSWER:

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number xxxxx.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

As we do with all histone modification antibodies in our lab, I used Active Motif's MODified Histone peptide array to test the specificity of the antibody before using it for a ChIP experiment. I've attached a powerpoint file with the associated data for this antibody as well as another antibody that I tested at the same time. For each antibody, there are two slides. I used Active Motif's free peptide array analysis software to do the data analysis. The first slide shows the graphical data of what modifications were hits on the array. The program presents the top 10 results. The second slide is an image of the peptide array with blue circles highlighting where the modifications that the antibody is supposed to specific for, were present on the array. As you can tell, the antibody lot for H3K27ac was not specific at all for H3K27ac.  As reference I also have attached the data from the Millipore H3K4me3 antibody that I tested. 

I have found in the literature that your antibody is commonly used for ChIP-sequencing experiments and I was hoping to do the same. 

Would you be able to send me other (different) lots to test specificity?  Let me know how we can proceed since we've ordered two vials of this antibody and it is not usable. If not, is there a way that our lab could be refunded?

ANSWER:

Thank you for contacting us.

I am sorry to hear of these issues regarding ab4729. I would be happy to send you a replacement lot. Should you find that this lot is not acceptable as well, I would be glad refund your original purchase. In order to proceed, could you please provide me with the Abcam order confirmation number for this purchase or with the PO number used?

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

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Dear xxxx,
the problem with the H3K27me,me2 and me3 is that they have opposite
meanings to the H3K27ac modification. I found that most antibodies
tested, also recognise to some degree those modifications. Now, if the
specificity is more than 75 fold higher for the H3K27ac modification,
it is considered a good antibody. If not, I would not use that
antidoby if I want to perform a chip-seq experiment because it is an
expensive tecnique and it may give rise to artefacts. H3K27me, me2 and
me3 are all repressive marks, associated to gene silencing. H3K27ac is
fairly poor characterised mark, but it is associated with an active
state.
This is why it is important to make sure that the antibody is specific.
I hope this information could be useful.
Regards,

ANSWER:

Thank you for providing this information to me. I am sorry about my late reply.

I will pass this information on to the lab so that they can take it into consideration when optimising the antibody. The lab are looking into using peptide arrays in order to validate the antibody which will hopefully be more accurate in determining the level of non-specificity. This is scheduled for June time. Once I receive information on how this progresses I will let you know.

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

I had a look to the data sheet that you sent me. I can see a reduction
in the signal when there is competition with a H3K27ac modified
histone, however the modification taken into account are very few. For
example, I found that most H3K27ac antibody also recognise H3K27me,me2
and me3. This modification has not been tested by your labs. Also,
from the western blot it is clear that the antibody also recognise
something else (probably histone 4) a part from histone 3. Obviously,
I am not able to quantify the intensity of the signal from the image,
but I would aim to follow the criteria used by Lieb. et al (attached)
and the histone 3 band from the figure does not seem 10 fold more
intense than the second band.
For these reasons, I am not thinking of buying the lot that you
suggested me. Thank you very much for all the effort and support though.

ANSWER:

I am sorry that you feel that way but I do understand.

We are hoping to start testing this antibody in peptide array in June time and hopefully I can then share the results with you.

May I just ask, we try to pick the blocking peptides we use in the cross reactivity western blot based on what researchers are generally concerned with (the ones that are going to interfere with the ChIP experiment). I am unfamiliar with the particular research area you are in but would you say that the H3K27me, me2and me3 are very important to you and other researchers? Are there any others you would suggest? I would be very interested to know as I will pass this on to the lab for them to consider.

I hope you have a nice weekend.

Thank you so much, Karin! All this support has been great!
Regards,

ANSWER:

Please find attached the batch testing data for lot number GR71158-2. Both the specificity and effectiveness in ChIP look good. I would say though that even though the specificity against the H3 acetyl K9 modification alsolooks good, we would expect some cross reactivity (as stated on the datasheet of ab4729) due to the high homology surrounding the modified residue.

If you would like to try this in your peptide array, I would suggest it may be worthwhile trying to use the conditions we use in the lab in order to perform such experiments.

1. We always use two concentration conditions, 2 ug/mL as you have been using, as well as 0.2 ug/mL. If a true non-specificcross reactionis observed thecross reactionproportion/percentage compared to the target modification will be the same. However, if thecross-reactivity is due to too much primary, the proportion/percentage will drop out and decrease at the lower primary concentration.

2. We use 5% BSA in TBST for blocking. We frequently find that BSA is a superior blocking agent to milk (which I suspect the Active motif blocking buffer is based on). I would suggest considering to use this. We have also found TBS to create less background compared to PBS.

3. We incubate the primary antibody for 2 hours at room temperature with agitation then wash with TBST:

2x5 min with agitation

2x10 minutes with agitation

4. We use a Alexa Fluor 647 labeled goat secondary antibody in a 1/10,000 dilution for 2 hours at room temperature with agitation. I donot know which secondary antibody you have been using but please be sure to not use it in too high a concentration. The wash steps above are then repeated.

5. As we use fluorescent reporters it is a little different to your data collection but I would suggest collecting different time points of exposure to analyse.

I hope this information has been of help. I would really be very interested to see how you get on with the new lot. If you do have any problems, please do let me know and I'll try to help as best I can.

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