Anti-ZAP70 (phospho Y292) antibody (ab12868)

thanks for the tech support. My jurkat cell line is constitutive Zap70 active and confirmed by my many previous data. I do think it is so critical as you have mentioned to my enquiry. The reason I could not detect the signal is obvious because of quality of this antibody. The past result is available upon request. I look forward to hearing from you again .

ANSWER:

Thank you again for your patience, I have just heard back from the originator of this antibody. Are you not seeing any signal at all? The originator said that it worked well for them on an endogenous system.

Also, ZAP-70 tyrosine 292 is the negative regulatory site that modulates the duration of activated TCR at the cell surface, unlike tyrosine 315 & 319 which one would expect to be phosphorylated in constitutively active ZAP-70 expressing cells. So you may want to try ab12869.

For both ab12868 and ab1269, it is suggested to use Jurkat cells treated with hydrogen peroxide as a positive control.

BATCH NUMBER 88930 ORDER NUMBER 67144

DESCRIPTION OF THE PROBLEM No signal only fussy background

SAMPLE Jurkat cell lysates

PRIMARY ANTIBODY 1ug/ml rabbit primary antibody in 3%BSA later 3ug/ml primary antibody in 3% BSA overnight Wash three times 5 min for each with 0.1%TBST

SECONDARY ANTIBODY Goat anti rabbit from BioRad 1:1500 dilution incubated for 1hour at room temperature. Wash 5X times 5mins for each

DETECTION METHOD ECL

POSITIVE AND NEGATIVE CONTROLS USED Jurkat cells

ANTIBODY STORAGE CONDITIONS -80

-80C

SAMPLE PREPARATION Cells were lysed with lysis buffer with fresh protease inhibibor. cells extract was mixed with 2X sample buffer with B-ME and boiled for 5Min

AMOUNT OF PROTEIN LOADED 30 ul of protein were loaded

ELECTROPHORESIS/GEL CONDITIONS in to 8% SDS PAGE

TRANSFER AND BLOCKING CONDITIONS Protein on the Gel was transfered on to nitrocellurose membrane.under 50 constant volts for 2 hours, and blocked with 3%BSA for 1hour.

HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes

WHAT STEPS HAVE YOU ALTERED? the second time the primary antibody was increased from 1ug/ml based on the specific sheet to 3ug/ml based on the tech support.

ADDITIONAL NOTES The film was exposed up to 20 mins

ANSWER:

Thank you for your enquiry and I'm sorry to hear that you are experiencing difficulty with ab12868. You mentioned that you are using Jurkat cell lysates; did you treat the lysates with hydrogen peroxide? Jurkat cells treated with hydrogen peroxide are recommended as a positive control. As you can see from the Western blot image on the online datasheet, in lane 1 with lysates prepared from Jurkat cells left untreated there is no signal, whereas there is a signal in lane 2 with lysates treated with hydrogen peroxide.

Please let me know if you have any additional questions.