Anti-ATR antibody (ab10312)

BATCH NUMBER 160963 ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM too many bands and not any specific

SAMPLE whole cell extracts (human glioblastoma cell lines)

PRIMARY ANTIBODY Abcam antiATR, rabbit, ab10312, lot160963, dilution 1/15000 in PBS, BSA 1%, 2hr Room Temp, several 15 min washes in PBS-tween

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED very good loading control (actin) so it is nt an extract or membrane or transfer or detection problem, but truely a problem of primiray Ab (long experience of western in the lab, main technic used)

ANTIBODY STORAGE CONDITIONS 4°C

SAMPLE PREPARATION standard (protease inhibitor cocktail-Roche, heated in 1X loading buffer (SDS, DTT, blue)

AMOUNT OF PROTEIN LOADED 60 µg

ELECTROPHORESIS/GEL CONDITIONS reducing, 10%

TRANSFER AND BLOCKING CONDITIONS ON transfer, TG iX, 15 % Ethanol, PVDF membrane, blocking 5% non fat milk

SECONDARY ANTIBODY antirabbit-HRP (jackson) 1/5000 in PBS-T, 1hr RT, several 15 min washes in PBS-tween

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 3 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? increase the dilution because background whorse at 1/5000 and 1/10000

ANSWER:

Thank you for taking the time to fill out the technical questionaire and send it in to us. I apologise for the delay in getting back to you. However, I have more details of the western blot testing procedure for this antibody from the laboratory which I hope will help.

This antibody can often recognise extra bands which are fragments of ATR, although this has yet to be tested on glioblastoma cells. If the small bands are more prevalent relative to the full-size ATR in this cell line, there may be a problem which we can resolve by optimisation.

1. The small bands may represent fragments of ATR. Ensure the lysate is fresh and correctly prepared. Ensure samples are adequately denatured and reduced.

2. The gels should be of lower percentage, 4 to 8%.

3. The primary antibody is at a high concentration which may lead to non specific binding. The data sheet suggests using a dilution of 1:15000 to 1:30000. There has been an Abreview submitted for this antibod in which the antibody was diluted 1:50000 with good results.

4. The amount of protein loaded is quite high. Try 20-30ug per lane.

5. You can try increasing the blocking incubation time to 2-3 hours.

When the antibody was tested the laboratory, they used used 5% Non-fat Dry Milk in TBS with 0.05% Tween to block the membrane. The same was used as diluent for the primary and secondary antibody.

I hope this is helpful information and that these suggestions will improve the results. Should you still obtain an unsatisfactory result after using these suggestions, then please do not hesitate to get back to me.

my Mphile project is to concentrate on an ATM/ATR DNA damage model, and i am thinking to order the ATM antibody [2B5] (ab4471) and ATR antibody (ab10312) that used in flow cytometry, western blot and immunohistochemistry from your company.in order to make sure the satisfy of my result the quality of these antibodies are very important, so there are few questions i would like to ask and would be greatful if you could reply me as well.

first, is that your ATR [2B5] antibody cross reacts with ATM (ATX08) antibody?

second, same as above is that your ATM cross reacts with ATR?

it is really important that there is no cross reaction between these two antibodies as i want to exam ATM and ATR separately.

third. does your ATM [2B5] antibody works on western blot?

forth, the western blot diagram on the datasheet of ATR [2b5], could u explain to us in details about this diagram please, as there are two bands on this diagram, is that means your ATM and ATR antibody are corss react with each other, if so we could not order it as we do not want the ATM and ATR antibody cross react.

please e mail us back as soon as possible.

look forward to hearing from u

ANSWER:

You recently enquired about our ATM and ATR antibodies and my colleague Karen has already sent you a reply regarding the antibodies ab2354 and ab4471.

I have also investigated your enquiry regarding ab10312. The antibody should not react with ATM owing to complete divergence of amino acid sequence between ATM and ATR at the site to which the epitope maps. There is no indication that the antibody crossreacts with ATM.

I hope this further information helps,