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DESCRIPTION OF THE PROBLEM No signal SAMPLE 1) Whole cell extract from Jurkat cells- 30ug 2) Immunoprecipitated material from Jurkat whole cell extract - the presence of USP25 was confirmed by mass spectrometry. PRIMARY ANTIBODY ab-5092 1:100 in 5% nonfat milk in PBS for 1h 3X Wash with PBST SECONDARY ANTIBODY donkey anty-goat IgG-HRP (Santa Cruzcat # 2033)1:5000 in 5% nonfat milk in PBS for 1h. 3X Wash with PBST DETECTION METHOD ECL Plus POSITIVE AND NEGATIVE CONTROLS USED N/A ANTIBODY STORAGE CONDITIONS stored @-20C SAMPLE PREPARATION Cell Lysis Buffer: 10% Glycerol; 20 mM Tis-Cl, pH8.0; 200 mM NaCl; 1 mM EDTA; 0.1% NP40 + Complete mini Protease Infibitor (Amerhsam) AMOUNT OF PROTEIN LOADED 30ug ELECTROPHORESIS/GEL CONDITIONS NuPAGE 4-12% Bis-Tris Gels (Invitrogen)with 1X MOPS Buffer TRANSFER AND BLOCKING CONDITIONS SureLock Transfer system (Invitrogen) for 1h @30W. Blocking 5% nonfat milk in PBS
Asked on Apr 28 2004
Thank you for your enquiry. We would be very grateful if you can clarify the followings: 1. Did you immunoprecipitate with this antibody? 2. Are you saying it works in IP but not on Western blot application? 3. Or did you IP with another antibody, show the IP contained USP25, then find our antibody gave nothing on western? We would like to emphasize that this is a Fast-Track antibody and it has only been tested for ELISA. We are looking forward to hearing from you soon.
Answered on May 04 2004