Anti-TRF1 antibody - ChIP Grade (ab1423)

I see a strong band at 50 kDa but only a very weak band at 60-65 kDa, where the antibody datasheet says this is observed. Other TRF1 antibody datasheets also show the protein at 60 kDa.

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 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.

follow up to previous correspondance:

1) Did the customer aliquot the vial upon receipt and uses a fresh aliquot every time?

-> yes!!

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.

-> whole cell extract. but I tested TRF2 of other company, I detected TRF2 very well with whole cell extract.

3) Were the protease inhibitors added freshly to the lysis buffer? Were samples kept on ice at all times?

-> of course! PIC was added and all samples kept on ice well.

4) Was the sample mixed with loading buffer and boiled?

-> yes

5) You say "amount protein loaded: 50ug" is this for the WB only or for the IP too?

-> No, I used 2mg for IP. Input was 50ug of protein.

6) Has the customer tried 5% BSA block?

-> yes, but same result.

7) has the customer tried a higher dilution with the IPed samples?

-> of course. I tried 1/500, 1/1000, 1/2000, 1/5000.

8)What is the buffer used to dilute the primary antibody and the secondary antibody?

-> 5% milk(5% BSA also) and TBST only.

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.

-> orc4 band was not faint. just scanner was bad.. and other our lab members see orc4 everyday. I think TRF1 antibody (delivered to me) has something wrong.

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.

-> but I detected well orc4(40kDa) and other proteins of low molecular (lower than TRF1) with 7% SDS gel.

also, I tried again with 12% gel. but result was bad..

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?

-> 1 tube : whole cell extracts(1mg, 2mg of protein) were incubate with protein A beads (G beads also) for pre-clearing.

2 tube : lysis buffer and TRF1 antibody (diluted 1/500,1/1000, 1/2000, 1/5000.) were incubate with protein A beads (G beads also) for TRF1 antibody can attach to beads.

... after that, pre-cleared cell extract of 1 tube was transfered to protein A beads attached TRF1 antibody of 2 tube and incubated for 2,3,4,5hrs and O/N.

ANSWER:

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,

Our end user claimed about ab1423.

She performed western blot , and IP, but she coull not detect band.

When she performed westernblot, she expected 60-65 KDa band according your data sheet.

But she could detect 75KDa band. Please confirm this.

And she performed IP, there was high back ground and no TRF1 band .

I attached claim report and data.

Check this.

ANSWER:

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,

Hi,

Thank you for your time and for all the information. My protocol is almost identical to this one that you gave me. I also tried two other protocols.

If it looked like I was getting something close to what I expect then I would persevere but none of the changes that I have tried have made any difference - I just see the diffuse staining throughout the nucleus and cytoplasm.

And so, I went to your website to go through the proceedings to return the product. However, when I clicked on the link to get the form in the 'return and replacement' policy under 'terms and conditions' the link does not take me to a form.

Can you help me with this?

Thank you,

ANSWER:

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.

Has the epitope been determined? What region of TRF1 does this antibody target?

ANSWER:

Thank you for your enquiry. Ab1423 was raised against the full length human TRF1 protein, so it recognizes the full length protein. If you have any more questions, please contact us again.