Anti-NQO1 antibody (ab2346)

I have problem with a particular batch of the anti-NQO1 antibody ab2346. The batch that work is 44897 whereas batch 105593 does not work when analyzed in parallel. It gives a high signal-to-noise in immunohistochemistry analysis. I do not believe that batch 105593 batch got spoiled during shipping since I have received it twice from you and both

are equally bad. Thus I'm interesting in batch 44897, would it possible to purchase that particular batch?? Best regards Staffan Bohm

ANSWER:

I'm very sorry to hear you are experiencing problems with lot 105593, we do not have any more vials of lot 44897 or 105593, and if you could provide me with the order details of the vials of lot 105593 I can certainly arrange replacement vials if you have purchased those in the last 90 days,

I look forward to hearing from you to arrange this,

Thank you for getting back to me so promptly regarding the NQO1 antibody. I originally purchased the antibody from Novus Biologicals. They suggested that I contact your company since you are the original seller. I also sent a copy of the Western Blot to Novus for them to review for a refund.

If you would like, I would gladly test the antibody for reactivity to mice since I do have liver cytosol from NQO1 null mice. At this point, I do not even have any left of the first aliquot we purchased. Please let me know if you can send me an aliquot of the new batch despite purchasing the original batch from Novus. I can provide you with shipping information.

ANSWER:

In a situation like this, you can either get a refund (from Novus and we in turn would refund them) or another batch. So I can directly send you another batch of ab2346 once it comes into stock. Please send me your shipping address, including phone number.

DESCRIPTION OF THE PROBLEM I ran liver cytosol from NQO1 knockout mice (obtained from the NIH) and had the same bands in the wild-type and knockout mice. This leads us to believe that we are not detecting mouse NQO1 using ab2346. Additionally, a prototypical inducer of NQO1 (BHA) was given to mice. Similar to the knockout mice, there was no difference in bands detected. While we are primarily interested in mouse NQO1, the antibody did recognize human recombinant NQO1.

SAMPLE CD-1 mouse liver cytosol, NQO1 knockout mouse liver cytosol, liver cytosol from BHA-treated mice

PRIMARY ANTIBODY ab2346 (anti-mouse NQO1) 2 ug/ml in TBS-T for 2 hours

SECONDARY ANTIBODY 1:30,000 anti-goat IgG (NB 710-H) for 20 minutes

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Positive control - human recombinant NQO1 Sigma Chemicals, D-1315 - WORKED Positive control - butylated hydroxyanisole (BHA) treated mice - DID NOT WORK Negative control - NQO1 knockout mouse liver cytosol from NIH - DID NOT WORK

ANTIBODY STORAGE CONDITIONS -20C

SAMPLE PREPARATION Samples prepared in sucrose-Tris buffer containing aprotonin and PMSF. Samples were boiled 3 minutes prior to electrophoresis

AMOUNT OF PROTEIN LOADED 50 ug protein/well for cytosol samples 4 ug protein/well for recombinant human NQO1 (Sigma Chemicals, D-1315)

ELECTROPHORESIS/GEL CONDITIONS SDS-PAGE 10% running/4% stacking gel

TRANSFER AND BLOCKING CONDITIONS Transfer in NOVEX system, overnight at 4 C onto PVDF membrane, transfer assessed by Coomassie staining at end of immunostaining, blocked in 1.5% fish gelatin as per Novus Biologicals

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? We have been using knockout liver and are NOT seeing any difference in bands compared to wildtype tissue suggesting that ab2346 is not detecting mouse NQO1

ANSWER:

I'm sorry to hear that you are having problems with this antibody. I have contacted the originator of ab2346 and there is a new batch of this antibody with better ELISA results than the previous batch which you received. The new batch has only been tested so far on human samples (human kidney and liver lysate) but is expected to cross-react with mouse due to sequence homology.

Would you like a replacement vial of the new batch free of charge? Did you order directly from Abcam? Please send me your Abcam order# or PO# and I can send you the replacement vial once it comes into stock.

Did you try this antibody for immunostaining assay in mouse tissues before?

ANSWER:

This antibody has only been tested on mouse liver lysates in Western blot.