Anti-ATR antibody [2B5] (ab4471)

I've tried a couple of the suggestions in your email, but I've still had no luck. Could you send me a replacement vial please, perhaps with a different batch number?

ANSWER:

I'm very sorry to hear the recent experiments did not give you satisfactory results.

I have arranged for the replacement vial to be sent to you, on order 189135 (a confirmation fax has been sent to this number: 01214158781); we have some in stock (of the same master stock but it will have a different batch as it was aliquoted at a different date) therefore it will be shipped to you this afternoon and you should get it tomorrow hopefully.

No one else has had problems with this antibody recently (one complaint in July) so I think the problem must have been with the vial you received, and the new vial should give you good results. Can you please let me know how you get on with the new vial?

BATCH NUMBER 10793 ORDER NUMBER MCA16126

DESCRIPTION OF THE PROBLEM Too many bands, no single band at 317kDa as shown on datasheet

SAMPLE HeLa cell extracts, separated into soluble, insoluble & total (see scanned blots attached)

PRIMARY ANTIBODY ATR 2B5 (ab4471), 1/1000 dilution in 5% milk in 1x TBS-T Incubation overnight @ 4 C Washed 6x in TBS-T

DETECTION METHOD ECL (Amersham)

POSITIVE AND NEGATIVE CONTROLS USED See scanned blots

ANTIBODY STORAGE CONDITIONS -20 C

SAMPLE PREPARATION TGN buffer for soluble proteins (50mM Tris pH 7.5, 150mM NaCl, 10% glyerol, 1% Tween-20, 0.2% NP-40) Urea buffer for insoluble & total proteins (50mM Tris pH 7.3, 9M urea, 150mM B-mercaptoethanol)

AMOUNT OF PROTEIN LOADED 50ug

ELECTROPHORESIS/GEL CONDITIONS 7% Tris-Acetate reducing gel

TRANSFER AND BLOCKING CONDITIONS running buffer = 1x Tris-Acetate Buffer transfer 6h @ 300mA blocking agent = 5% milk in 1x TBS

SECONDARY ANTIBODY Secondary anti-mouse Incubation 2h @ RT Washed 6x in TBS-T

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

WHAT STEPS HAVE YOU ALTERED? N/A

ADDITIONAL NOTES I have attached the results from the Western. I have three Hela lysate samples, lane 1 is insoluble proteins, lane 2 is soluble proteins, lane 3 is total proteins. I stripped the membrane and re-probed with a p300 antibody which recognises CBP/p300 at 300kDa so 300k is marked along with the 200k band from the ladder.

ANSWER:

My sincere apologies for the delay in resolving this matter, I have been ill the last few days and was still waiting to hear back from the source of ab4471 for some feedback. They replied to me the following comments: "The only suggestions that I could add are obvious and I am sure they have already been suggested to the customer: the primary antibody might be too concentrated - try decreasing the concentration of the antibody and/or decrease incubation time. Running a no primary control might also help determine if the secondary antibody could be causing a problem."

I would be happy to offer you a replacement vial or refund if you have tried the following tips and still had problems, please just let me know which you would prefer,

...hopefully u still remember that i have explained to u that the ATR i got last time was not working at all in immunocytochemistry, in fact it worked the first time when i got it, so i just wandering can the un-worked one replaced...u ask me to give u thte deliver number, but have been talking to one of the secretary whose in charge of the order and she said that she didn't have any deliver invoice for ATR, but she gave the order number which is 98062373, this was ordered on 12/04/06... and the invoice number for this order is 1024762. hopefully this will help... thanks for your attention.

ANSWER:

Thank you for your e-mail and your order details. I have arranged for a replacement vial to be sent to you; this is placed on order 161886 and should be dispatched today.

Could you please let me know if you are satisfied with the replacement vial?