Anti-ECSIT antibody (ab21288)

Dear Provider,

we are extensively using your Anti-ECSIT antibody (ab21288), which is giving us high quality results. Nevertheless, we are also experiencing some intriguing results, which may account for post-translational modifications that ECSIT might undergo. Unfortunately, since the sequence of the antibody is not provided (just that is 14 amino acids near the C-terminus), we cannot prove our hypotheses. We would be grateful if you can provide us this information in order to complete our experiments.

We thank you very much in advance.
Best,

ANSWER:

Thank you for your enquiry and your interest.

I regret to inform you that the immunogen sequence is commercially sensitive information and we are not allowed to release it. However, it is between residues 301 and 350 aa accession number NP_057665.

Since antibody is a polyclonal antibody, it recognizes different epitopes so epitope mapping is not performed.

If you need any further assistance, please do not hesitate to contact me.

Please see reply from Nezira below. regards

--- Hi Michelle, i have just received your email about the antibody from abcam, I am sorry but I dont have the lot number from the previous vial as i bought it about 1 year ago and have already used it all. The samples that i tested it on would have been the same because i am still using the same plasmids so the myc tag would have not affected it. I would be happy to take the replacement as they are still the only supplier for this antibody and i am eager to continue with the experiments.I would also be happy to receive any help on how to improve the signal because even with the previous vial that i bought i did not get the same band as they have on the data sheet which comes with the antibody.I am mainly working on the HEK 293 cells but am also looking at PBMCs and CD14positive and negative cells.

Thanks Michelle for your help.

ANSWER:

Thank you very much for your reply and Nezira's.

At this stage I think it would be worth looking at Nezira's protocol in detail in light of the fact that the first lot did not give her the results expected. Can you please ask Nezira to click on the link below and fill in our online questionnaire for WB, we will then be able to review all her data and understand the source of the problem. I would already recommend to run a positive control of human heart lysate to make sure that the problem is not due to her sample preparation.

http://www.abcam.com/index.html?section=western&pageconfig=technical&intAbID=21288&mode=questionaire

We look forward to receiving this information to resolve this matter, thank you both in advance,

BATCH NUMBER 130274 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM Wrong band. It detects a band that looks like it is the correct size, but when truncation mutants of ECSIT were made the band does not change size.

SAMPLE Recombinant protein

PRIMARY ANTIBODY Abcam ab21288, Diluted 1:1000 in TBST containing 2% BSA 0.01% NaN3. Incubated overnight at 4 degrees C. Followed by 3 quick washes, 3 15-min washes and 3 quick washes.

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Negative: Recombinant GFP and untransfected cells. Positive: Recombinant tagged ECSIT (of various lengths due to ECSIT truncation mutatants)

ANTIBODY STORAGE CONDITIONS 4 Degrees C, as it arrived

SAMPLE PREPARATION SDS Loading Buffer, 3 min at 95 degrees C

AMOUNT OF PROTEIN LOADED 1 well of 24-well plate expressing protein. This is what I generally use for western blotting and works well for other combinations of recombinant proteins and respective antibodies.

ELECTROPHORESIS/GEL CONDITIONS SDS PAGE, 4-12% Novex Bis Tris

TRANSFER AND BLOCKING CONDITIONS For transfer: Invitrogen NuPage Transfer For Blocking: TBS-Tween (0.15M NaCl, 0.1 M Tris HCl pH7.4, 0.1% Tween 20) with 3% milk

SECONDARY ANTIBODY [a competitor] goat anti-rabbit HRP conjugated 1:5000 Incubated 2 hours at 25 degrees C. Followed by 3 quick washes, 3 15-min washes and 3 quick washes.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 5 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Different cell lysates from independent transfections. Used fresh primary and secondary antibody dilutions every time.

ADDITIONAL NOTES Uncertain which of the two is the order number: S102487 or 4500103431

ANSWER:

Thank you again for your enquiry.

I spoke to a colleague of mine who also agreed that you may be detecting the endogenous protein. Did you cleave the tags? The tags may be adding the additional kDa’s to your bands. Another possibility is that differences in the size could be negligible enough that they do not register on your gel. There just is not enough information on your truncation methods to determine where the problem lies. Where is your truncated sample cleaved? The immunogen is within residues 301-350and I hope this information can help you.

Please do not hesitate to contact us if you need any more advice or information.