Anti-TRF1抗体(ab1423)

Thank you for your reply.

Our datasheet for this antibody does not suggest any specific ICC protocol, as a general ICC protocol should work with this antibody. The protocol stated on the datasheet belongs to a customer’s review who submitted an Abreview for this antibody in that application, and that can actually serve as guidance for other researches.

I understand, however, that this antibody should have given the expected results under the conditions you have used it, and it may well be that you have received a faulty vial.

If the antibody is still under the 6 months guarantee I will be happy to either send you a replacement for the same or a different antibody, or, as you mentioned in your last email, refund the product’s price.

Could you please send me your order number to be able to raise the replacement / refund?

Thank you very much for your cooperation. I look forward to receiving your reply with further details.

Thank you for contacting us and bringing this to our attention. The switch to PFA fixation and detergent permeabilization instead of methanol fixation that I mentioned in our conversation may help, as that is what has worked for customers in the past.

Please let us know if you continue to have difficulty.

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 1016117.

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.

Thank you for this information, it is very useful and I can now really understand the protocol and the optimisation steps. I think the WB protocol looks very good (apart from my suggestion of running a high percentage gel), the IP protocol we suggest is very different but the fact the customer has a problem in WB means the problem is not related to the IP protocol. I understand that the customer can detect TRF2 in whole cell extracts, but it is possible that the TRF1 levels are much lower and a nuclear extraction would concentrate the protein A/G-sepharose beads for 4 hours and finally boiling the sample . If you can provide me with the purchase order or Abcam order number I can send you a replacement antibody to try, but I would strongly recommend nuclear extraction to maximise the amount of TRF1 protein loaded on the gel. I would also suggest changes to the IP protocol: incubate the cell extracts with the antibody overnight (try 5ug antibody per 500ug lysate), then the next morning add the protein A sepharose beads, incubate together for 4hrs, then wash the beads and boil, separating the protein from the antibody and from the beads. Please do not hesitate to contact us for further details of our recommended IP protocol. I look forward to hearing from you,

I have carefully looked at the protocol and unfortunately need more information to understand the problem, I am sorry for the delay. The antibody has not been tested on HeLa cells, it does cross react with human but it is known that cancer cells can be very different morphologically and biochemically, this may explain why the signal is low. 1) Did the customer aliquot the vial upon receipt and uses a fresh aliquot every time? 2) Is the HeLa cell extract a nuclear extract? The protein is a nuclear protein and absence in the WB could be due to the low amount of TRF1 in the whole cell lysate. 3) Were the protease inhibitors added freshly to the lysis buffer? Were samples kept on ice at all times? 4) Was the sample mixed with loading buffer and boiled? 5) You say "amount protein loaded: 50ug" is this for the WB only or for the IP too? 6) Has the customer tried 5% BSA block? 7) has the customer tried a higher dilution with the IPed samples? 8)What is the buffer used to dilute the primary antibody and the secondary antibody? 9) The WB image you provide says "TRF1 is not detected but orc4 is detected well". I see a faint band around the 55kDa mark which could be TRF1. The Swiss Prot database indicates that the MW of TRF1 is around 50kDa. The customer is happy with the faint band of orc4 but I would disagree and optimise the protocol for both antibodies. 10) When investigating low molecular weight proteins the customer should use high percentage gels, use of a 7% gel means the bands are at the bottom of the gel and not well separated. 11) for investigating the problem with the IP, I would need a detailed protocol of the IP. I suggest we first concentrate on the WB to find out if the antibody works. I believe the faint 50kDa band might be the TRF1 but an optimised protocol is necessary to get a clean band. The background from the IP samples could be due to the blocking agent used, the type of protein A or G beads used for the IP and other factors. Has the customer much experience of IP? I look forward to hearing from you, Thank you for your patience,

I'm sorry to hear that this antibody is not working out for you. If you send me the Abcam order number or purchase order number that was used for your order for ab1423, I can give you a refund. There is no need to return the product to us.