Anti-BCL2L1 antibody (ab2568)

DESCRIPTION OF THE PROBLEM Non-specific band The Bcl-xl antibody ab2586 recognises strongly (only) a band of 48 kDa on Western blots of total cell extracts of rat hepatocytes (in milk, TBS+0.1% Tween-20 or the same without the Tween). We used the recommended dilution of it. Any ideas why/ how to correct this?

SAMPLE total cell extracts of rat hepatocytes

PRIMARY ANTIBODY Ab2568 1 ug/ml

DETECTION METHOD ECL

ANTIBODY STORAGE CONDITIONS -20 C, thawed for the first time.

SAMPLE PREPARATION homogenised on ice, boiled in laemmli

AMOUNT OF PROTEIN LOADED 100 ug

ELECTROPHORESIS/GEL CONDITIONS acrylamide/bis 12/0.5

TRANSFER AND BLOCKING CONDITIONS semi dry blotting, 5 % skimmed milk for 1 h, primary and secondary Ab in milk

SECONDARY ANTIBODY anti rabbit-HRP BioRad, 1:5000

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? TBS pH 7.6 with or without 0.1 % Tween-20

ANSWER:

Thank you for your enquiry.

I am sorry to hear that you have been having difficulties with this antibody. We have received some excellent feedback for this antibody in the form of customer "Abreviews". They all detail a strong band of approximately 30KDa. It is puzzling to me why this antibody detects a band of this size as the approach that you are using is one that I would recommend.

To further optimise this antibody I would recommend that you perform a no-primary control experiment to determine whether this band is the result of the primary antibody. I would also like to recommend that you reduce the amount of protein that you have been using from 100ug to 20-30ug as a mass of this amount may be increasing the degree of non specificity.

It may also be useful to try an alternative blocking agent such as BSA. Our lab team frequently find that a change to the blocking agent results in improved more specific results.

Should you continue to have difficulties please get back in touch with me.

I am looking for an anti-Bcl-XL antibody that works in immunoprecipitation, I was thinking about the product ab3193 or ab2568, since both should work in immunoprecipitation, do you have any reference of published papers where these products have been used in immunoprecipitation assays?

ANSWER:

Thank you for your enquiry. All the information that we have regarding these two antibodies is located on the online datasheets, including references that we are currently aware of. If you have any more questions please contact us again.

Is the immunogen the whole BclXL or a fragment of it? I need a polyclonal ab against the whole folded Bcl XL.

ANSWER:

Ab2568 is against a peptide. However, ab2568 actually utilizes the whole protein as immunogen.

Concerning ab2568: Has anyone tried this antibody in immunohistochemical staining (frozen or parafin)? We are using porcine/pig tissue. Thanks.

ANSWER:

To our knowledge, this antibody has yet to be tested in this application. All tested applications are specified on Abcam product datasheets. If you decide to go ahead and purchase this product, please let us know how you get on and in return we will forward a reward of your choice, typically an Amazon gift voucher.

Has the ab2568 antibody been tested on mice through immunohistochemistry (particularly on cryostat sections)? I would like to know any results and dilutions.

ANSWER:

According to the datasheet, this antibody has not been tested for IHC application. However, it is suitable for IP which suggests that this product recognizes the native form of the protein. Should you decide to go ahead and test this antibody, please let us know how you get on, and in return we will forward a USD15/EUR15/GBP10 Amazon gift voucher.