Anti-cIAP2 antibody (ab2540)

ab2540 lot # 61468

The problem I am having with ab2540 is that it does not appear to detect cIAP2 in the mouse. Using total cell lysate from MCF10A (human mammary epithelial) cells as a positive control ab2540 detects a single band at approximately 65 kDa that is likely to represent cIAP2 (see attatched Figure 1). However, no equivalent band is detected in either the FSK7 (mouse mammary epithelial) or MEF (mouse embryonic fibroblast) lanes.

The left hand side of Figure 1 represents protein extracted from mouse tissue by homogenising in RIPA buffer containing protease and phosphatase inhibitors. Again, no band appears likely to represent mouse cIAP2. The expression of cIAP2 in the tissue samples used for the western blot has been confirmed by reverse transcriptase PCR.

Western Blot Protocol:

Ab2540 was raised in the rabbit and 'secondary only' control blots have been performed to demonstrate that any bands visible are due to the primary antibody.

Approximately 20 µg of protein is loaded in each lane of an 8% SDS-PAGE gel and separated under reducing conditions. Transfer to a nitrocellulose membrane is achieved at 100 V for 90 minutes. The membrane is then blocked using 5 % non-fat milk/ 0.1% Tween in PBS either for 2 hours at room temp or overnight at 4 degrees.

Primary antibody solutions of between 1 and 3 µg/ml were tested with 2 µg/ml been the optimum. The blot was incubated in primary antibody for 2 hours at room temperature. Between primary and secondary antibody the membrane is washed 4 x 5 minutes with PBS/0.1 % Tween.

The secondary antibody was HRP-conjugated Donkey anti-rabbit IgG from Jackson labs, which is used at 1:10,000 and incubated with the blot for 1 hr. Before detection of bound secondary antibody the 4 x 5 minute washes are repeated and supplemented with a futher 5 minutes in PBS alone.

Detection is achieved using ECL detection kit (Amersham) and Kodak Biomax XAR film.

Regards

Tom Owens

ANSWER:

Thank you for taking the time to submit your concerns and protocol, having now read through the information that you have sent me. There are a few suggestions that may be made.

1) From the gel the bands that you are observing in lanes two and three of the "mouse tissue" seem to correlate with the bands that you observe in lane 14 and 15 that you have identified as the target?

Re-run the gel on a ten well gel. Run the sample with a molecular weight ladder, this is currently not present (unless the ladder has been chopped off?). Wider wells will enable you to view the gel more clearly. Run these lanes against a positive control, previous customers have detected single bands using HCT116 lysate

2) What loading control have you used to ensure that all the wells have been loaded evenly?

2) What is the tissue are you using that you refer to as "mouse tissue"? To what extent is the target present in this tissue?

If you are still experiencing problems than please get back in touch with us. Good luck with your research.

Do you have any western blot data that show ab2540 detecting mouse cIAP-2, as the immunogen (aa 507-524 of human cIAP-2) shares only 55% identity with the mouse protein (accession NP_031490)?

ANSWER:

Thank you for your email. This antibody has been tested for cross-reactivity with both human and mouse, however we do not have any images available. If you have any more questions, please contact us again.

What is the original source of ab2540?

ANSWER:

We produce some of our antibodies in-house and others are sourced from suppliers whose products we believe will be of interest to our customers. This product falls into the latter category. The identity of our suppliers remains commercially sensitive information.

I would like to know if you have any additional information available regarding your antibody ab2540 against cIAP2/HIAP1, specifically: The immunogen: full length; peptide; C/N-terminal etc. (we already have antibodies against cIAP2 from R&D Systems) Whether he antibody is suitable for RAT.

ANSWER:

The immunogen is 507-524 of human cIAP-2. The antibody has not been tested with rat sample, and has only been tested for application in Western blotting. Please refer to the online datasheet for ab2540 for any references that we have.