Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)

Dear
Our customer’s schedule is delayed because of this problem.
He is sure that this antibody has problem because it was not only detected multi-bands but also shown bands in recombinant Histone H3(Millipore) which non-methylated protein.
Please check this issue as soon as possible.
Best regards

ANSWER:

Thank you for your patience.

After reviewing this case with my lab colleagues they have confirmed this antibody has not been tested with the recombinant protein but instead we test with CTH nuclear prep with a variety of modified peptides plus unmodified. Our WB results show good blocking with the H3 mono methyl K4 peptide and no blocking with the non mod or other histone modifications.

The only difference they noticed is the blocking agent. Whereas we use 5% BSA in the lab, your protocol used milk. We are not sure whether this could make any difference to your protocol, so I would be happy to send a replacement for the same antibody from a different lot or a credit note instead.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

multi-bands Our customer conducted WB to check specificity of the antibody before doing ChIP experiment. The data was shown non-specific multi-bands in the OVCAR3 sample. Even this antibody detected recombinant Histone H3.

ANSWER:

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

In order to better understand the problem, I would like to send you a questionnaire with some additional questions about the protocol used, as well as the order details. All this information is crucial to help us understand the possible causes of the problem.


Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results.

I look forward to receiving your reply. Please do not hesitate to contact us if you need any more advice or information.

Dear abcam technical support team:
This customer already purchase ab8895, ab32521, ab32356, ab12079, and conducted WB with human sample, but now he would likes to use these antibodies and peptides to test in ELISA assay with human sample, would you please help this customer to create a discount code?

ANSWER:

Thank you for contacting us.

ab71998 and ab71999 have already beentested in ELISA.

For the other antibodies, I am sorry we cannot issue testing discount codes for antibodies that have already been purchased.A testing discountcode must be issued before the order is placed. However, we like to encourage all of our customers to submit an Abreview via the online product datasheet. We always appreciate customer feedback, whether positive or negative, and we make all product information available to researchers. Plus, each Abreview earns Abpoints that can be used for discounts on future purchases.

To find out more about our Abreview system, please see the following link:

http://www.abcam.com/abreviews

I hope this information is neverthelesshelpful. Please do not hesitate to contact us again with any other questions.

Hi

THank you for your reply. But unfortunately you misunderstand me. I am looking for positive control of H3K4 mono, di- and tri-methylatied H3 (full size protein) on WB. These positive control have to show the same MW as unmodified H3 protein when they are loaded on the gel. You gave me information of peptide which is useful for absorption of antigen. These peptide are not full size of H3 protein. Unfortunately this is not what I wanted. Do you have any full size of H3K4 mono-, di- and tri methylated histone 3 which I can load on the gel as positive control. I am looking for your reply. thanks!!

ANSWER:

Thank you for contacting us. I apologize for the confusion in our earlier correspondence.

The molecular weights of the different methylation states be extrememly difficult to distinguish as that type of modification creates a 14Dalton mass change.Currently, we do not offer full length methylated H3K4 proteins. We do have anEpiSeeker Histone Extraction Kit (ab113476) whichprovides a simple and selective method for extracting histone proteins in just 1 hour. When used in conjunction with our peptides you can be confident that the band you are seeing is indeed specific to the mono- di- or tri- methylayed H3K4 protein.

More information about ourEpiSeeker Histone Extraction Kit (ab113476) is available here:

http://www.abcam.com/EpiSeeker-Histone-Extraction-Kit-ab113476.html



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

Hi

We have your antibodies against Histone H3K4 methylation (ab8895 for monomethylation and ab8580 for trimethylation). I would like to see their specificity on WB. Do you have any WB data loading Histon H3 unmodified, monomethylated H3K4, dimethyated H3K4 and trimethylated H3K4 and then stained by ab8895 or ab8580 ? Also do you have any control peptide/protein applicable for WB of H3K4 methylation WB ? They should be full size of monomethylated H3K4, dimethylated H3K4, trimethylated H3K4 and unmodified H3. I am looking forward to your reply. thanks!!

ANSWER:

Thank you for contacting us.


Peptides are available for both of these products. The peptide for ab8895,Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade, is product number ab1340 (http://www.abcam.com/histone-h3-peptide-mono-methyl-k4-ab1340.html).The peptide for ab8580,Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade, is ab1342 (http://www.abcam.com/histone-h3-peptide-tri-methyl-k4-ab1342.html). These are the exact peptides used to create the antibodies. Each peptide used in the testing assay is available as well. (http://www.abcam.com/index.html?datasheet=1340,http://www.abcam.com/index.html?datasheet=7768),http://www.abcam.com/index.html?datasheet=1342,http://www.abcam.com/index.html?datasheet=1771,http://www.abcam.com/index.html?datasheet=1780,http://www.abcam.com/index.html?datasheet=2903.

These products are tested by peptide assay. Our data on western blot is displayed on the datasheet for each product. For ab8895 our test show itspecific for mono-methylated Lysine 4 of histone H3 and does not recognize di- or tri-methyl Lysine 4 nor methylation at Lysine 9. In the image on the datasheet, this is shown in lane 2where the activity of the antibody is specifically blocked by the addition of the immunizing peptide (ab1340) [http://www.abcam.com/Histone-H3-mono-methyl-K4-antibody-ChIP-Grade-ab8895.html#ab8895_6.gif].

Further,ab8580 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - tri methyl K4 peptide (ab1342), indicating that this antibody specifically recognises the Histone H3 - tri methyl K4 modification [http://www.abcam.com/Histone-H3-tri-methyl-K4-antibody-ChIP-Grade-ab8580.html#Histone-H3-Primary-antibodies-ab8580-153.jpg.]

Each of these products are thoroughly characterized and published. There are links to specific publications and th Abreviews from our customers on each datasheet.

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