Anti-RUNX3 antibody - ChIP Grade (ab11905)

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED --

DESCRIPTION OF THE PROBLEM target band shape is not good and more bands and detected markers.

SAMPLE U937 cell extract

PRIMARY ANTIBODY abcam/rabbit/5% blocking solution/1:1000/ overnight/ TBST wash 15min 5 times

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Use positive control SNU16 and U937 // negative 293T

ANTIBODY STORAGE CONDITIONS -20 degree

SAMPLE PREPARATION Sigma M lysis buffer / roche protease inhibitor cocktail/ heating 100 degree 5min

AMOUNT OF PROTEIN LOADED First 20ug and antibody detected many bands so diluted sample 1/10, 1/50/ 1/200

ELECTROPHORESIS/GEL CONDITIONS 10% gel, reducing gel

TRANSFER AND BLOCKING CONDITIONS Semi dry transfer 40min/ 5% skim milk blocking

SECONDARY ANTIBODY santacruz/5% blocking solution/1:10000/ 1hour RT

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? Antibody dilution and sample dilution and blocking solution percentage

ADDITIONAL NOTES I want to know the concentration of this antibody, ug/ul.

ANSWER:

Thank you for taking the time to submit these comments. It looks to me from the gel that the gel has not run out properly. Could you please enquire with the customer as to how the gel was made and how long the running gel and stacking gel were left to polymerise for prior to use?

Smeared banding such as that shown can be due to running the gel at too high a voltage. Since the gel can loose rigidity creating band smearing. Under what conditions was the gel ran? I suspect that reducing the voltage or running the gel in the cold room would reduce the observed smearing.

I look forward to hearing from you again shortly.

BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER 210605

DESCRIPTION OF THE PROBLEM No specific signal, often nothing at all.

SAMPLE Human T cells as well as PBMC depleted of monocytes and NK cells

PRIMARY ANTIBODY ab11905, abcam, 1:1000. Incubation from 2 hr RT to overnight 4 degrees. 2 hr wash before addition of secondary.

DETECTION METHOD ECL plus

POSITIVE AND NEGATIVE CONTROLS USED Positive control: Jurkat lysate. No negative control

ANTIBODY STORAGE CONDITIONS storage -20 degrees, aliquoted in two lots of 25 ?l

SAMPLE PREPARATION lysed samples (buffer: 9 ml 5% triton x-100 in Tris HCL, 9g/l NaCl + 1 ml 0.5 M EDTA + complete protease inhibitor tablet (ROCHE)) mixed 1:1 with l?mmli buffer.

AMOUNT OF PROTEIN LOADED Varied, sometimes different amounts of same sample used on 1 blot.

ELECTROPHORESIS/GEL CONDITIONS 4-12% Nupage gel (Invitrogen), MOPS or MES buffer

TRANSFER AND BLOCKING CONDITIONS Transfer 3 hrs, 4 degrees in transfer buffer (Invitrogen), 10% Methanol., blocked with 1 % BSA, 1 hr.

SECONDARY ANTIBODY anti-rabbit-HRP, amaxa. 1:7000, 2 hr. wash two hr before development.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 4 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? Incubation time and temperature, amount of sample, type of cell lysate, development time

ADDITIONAL NOTES About purchase order number: The antibody was ordered by our technician, together with anti-RUNX1 (PON zumkehr210605. I have used the anti-RUNX1 antibody (ab11903) to satisfaction on the same samples and even blots as those used for this anti-RUNX3 antibody, also at a dilution of 1:1000 and with the same reagents. The much smaller amount of anti-RUNX3 supplied did not allow for dilutions less than 1:1000, without skyrocketing costs.

ANSWER:

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with ab11905. For Western blotting, we do suggest using this antibody at 1:500 - 1:1000 and so I do suggest trying ab11905 at 1:500 with incubation overnight at 4C. We also suggest using Raji nuclear extract as a positive control.

I hope this helps. If you need additional assistance, then please contact us again.