Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)

Customer called as they were seeing no bands using this product.

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.

To whom it may concern,

Thank you very much for all your help in this matter. It is great working with a company that cares about customer concerns.
If possible could be be refunded for either one of the two we purchased, and for the other exchange it for ab23366, a mono/dimethyl lysine antibody.

I am not sure of the order # or date as our purchasing office has closed for the day. If you need more information please let me know and I will talk with them tomorrow. Otherwise, I hope this information is sufficient.

Thank you again for all your time and help.

ANSWER:

As both antibodies failed to work in western blot, I have sent you ab23366 as a free of charge replacement for ab7315, your new order number is ******** andI have also credited the cost of ab412, your credit note number is **********.

I am sorry about the problems you had with these antibodies and I hope that ab23366 works out for you. If there is anything else I can do, please let me know.

Hope you have a good evening.

To whom it may concern,

Thank you again for your reply. I will try to provide you with the answers to the questions.

Question 1. see figure below. Our lab has done several MYC IPs and have IB with MYC antibodies to confirm the presence of MYC. In the figure I attached (most recent IP) I IP MYC using MYC C-33 and IB with a acetylated MYC antibody. I further stripped and re-probed the blot with MYC N262 to further confirm the presence of MYC in the IP

ANSWER:

I really appreciate you sending that further data.

Since it is clear that ab412 and ab7315 are not detecting your protein of interest in your western blot and under our Abpromise, we guarantee that both should work, then I would be very happy to either send you replacement antibodies or to process a refund.

Please let me know how you would like to proceed and if you would like to receive replacements antibodies, let me know the catalogue numbers of those you would like. Could you also provided me with the original order details for these antibodies (either order #, purchasing agent name, date of purchase) so that I can complete your request.

Thank you once again for your patience and I look forward to your reply.

To whom it may concern,

Thank you again for the fast reply. Here is the information you are asking for:

1. Yes, two different MYC antibodies for IP and a MAX antibody for a MYC co-iP
2. Myc band is typically seen about 62 to 65 kDa. For IP I run the gel much longer to avoid the MYC band from NOT separating from IgG band.
3. yes, we would like to only see bands in IP lane and not the control lane.


Thank you very much again for all your help and time.

ANSWER:

Thank you for answering those questions and your continued patience.

Firstly, I would just like to reiterate that both ab412 and ab7315 are guaranteed to work in IP and so if we cannot determine what the problem is, then I will be happy to replace or refund these antibodies.

Having said that, I do have a few more questions/suggestions:

1 - Have you confirmed that the Myc antibodies are actually IP'ing your sample of interest. You could determine this, buy using the Myc N262 to IP and then blot with MycC33.

2 - Your input sample (you load 20ug?), does give a signal for your protein of interest, but have you tried starting with more total protein. I ask this as although you are using 20ug of total protein but increasing that amount (to say 40ug) you may be able to get more of your protein of interest to actually be IP'd and so get a signal using the antibodies.

I look forward to your reply and helping you resolve this issue.

Thank you very much for your reply and your concern and any help you may be able to provide in exchanging or providing the early stated new methyl antibody.
Here is the information you asked for:
Samples: HeLa lysates made from 400mM NaCl lysis buffer.
Application of abcam antibodies: detection of a PTM (methylation) on the MYC protein.
Brief protocol:
block nitrocellulose membrane after transfer with 6% NFD milk for 30m.
1. incubate in primary antibody (either ab412, 1:1K or ab7315 1:1K in +4/ON)
2. Wash away excess primary with TBST X 3, 5m each wash
3. incubate in secondary HRP linked antibodies (1h for normal secondary 1:5K, 30min for true blot 1:5K + dark)
4. wash away excess secondary antibody with TBST X3, 5m each wash.
5. use ECL X1 min, and expose to x-ray film.

ANSWER:

Thank you for providing that extra information.

Just to make sure that I understand your western blots:

1 - You are using a Myc antibody to do the IP, correct?

2 - What is the size of your band of interest? As you are seeing a band about ˜60kDa in your inout lanes and also seeing the same size band in your control and IP lane.

3 - Are you only expecting to see bands in your IP lane, NOT your control IgG lanes?

Thank you for your patience in this, I just want to make sure that I understand the problems that you are having, so that I can give you the best advice or send you the appropriate antibody.

I look forward to your reply.