Anti-Bag3 antibody (ab5898)

BATCH NUMBER 78702

DESCRIPTION OF THE PROBLEM High Background on western blot and multiple bands

SAMPLE 40 ug whole cell lysate of human breast cancer cells MDA435 overexpressing different Bag-3 constructs

PRIMARY ANTIBODY TBST dilution buffer; dilutions of 1:200, with incubation for 1 hr at 4C

SECONDARY ANTIBODY 1:5000 in TBST for 1 hr at 4C (this dilution has been used successfully before)

DETECTION METHOD Chemiluminescent

POSITIVE AND NEGATIVE CONTROLS USED yes, see above- signal may be positive but hard to tell with background Negative control also had high background so hard to tell difference with high background

SAMPLE PREPARATION Standard lysis buffer as described in Maniatis, et al with Tris-HCl, NP40, at pH 7.9

TRANSFER AND BLOCKING CONDITIONS 4-12% Bis-Tris gel from Invitrogen; transferred to PVDF membrane for 1 hr and blocked for 1 hr with TBS-Tween (0.05%)

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 HAVE YOU RUN A "NO PRIMARY" CONTROL? No

WHAT STEPS HAVE YOU ALTERED? Tried to increase the stringency of wash buffer using TBST-H2O (v/v 50%) to water but background remains

ADDITIONAL NOTES Customer thinks the fact that you see a band at 80 kDa is correct due to phosphorylation of Bag-3. I have sent the high background notes on your site. Please review notes above and let me know suggestions plus the amount of protein loaded and the western dilution you used. Thanks.

ANSWER:

Thank you for providing more information about your protocol.

We would like to emphasize that this is a Fast-Track antibody and not fully characterized yet. Please take a look at the Terms and Conditions for Fast-Track products.

We cannot guarantee that the antibody will work in any application other than ELISA against the immunizing peptide and therefore we are unable to offer a refund if the antibody does not work in your application.

However, Fast-Track antibodies are offered at a reduced price

Our preliminary results show an approx 80 kDa band in HeLa and 293 lysates at 1ug of primary on Western blot application. Please note that currently we cannot find an explanation in the literature for the band we observe given the predicted size of approx. 67Kda according to NP_004272.

In order to be able to reduce the high background, we would suggest loading 20 or 30 ug protein per lane and/or dilute the antibody a bit further down (i.e. 1/500 or 1/1000).

We would suggest perhaps submitting a review on-line. In return we will forward a USD15/EUR15/GBP10 Amazon gift voucher.

We hope this will help.