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BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM No CCR5 band, only background bands. SAMPLE Human sample from blood derived Mature Dendritic cells. Purified protein using Pierce M-PER reagent. PRIMARY ANTIBODY Primary Antibody: Abcam rabbit polyclonal CCR5 at 1:1000, diluted with 3:1 with TBS-T and NAP-Blocker and incubated at 4C overnight. Wash 30 minutes with TBS-T Attempted second time at advice of abcam tech, using 5:1 dilution of TBS-T and NAP-blocker. Wash 30 minutes with TBS-T DETECTION METHOD Detected with 45 minute step of Invitrogen Streptavadin 1:2500, then 30 minute wash. Developed with Pierce NBT-BCIP solution at RT until bands develop. POSITIVE AND NEGATIVE CONTROLS USED None ANTIBODY STORAGE CONDITIONS Stored at +4C SAMPLE PREPARATION Used 1:1 Laemmli Sample Buffer that has 5% B-MERC. Heated sample for 5 minutes at 95C. AMOUNT OF PROTEIN LOADED 30 micrograms per lane ELECTROPHORESIS/GEL CONDITIONS GeneMate Express Polyacrylamide Mini-gels. 4-20% Express gels TRANSFER AND BLOCKING CONDITIONS Transfer: Transfered at 25V for 100 minutes onto PVDF membrane. Blocker: G-Biosciences NAP-Blocker 1:1 with TBS-Tween. Blocked for 30 minutes at RT. SECONDARY ANTIBODY [another company] goat anti-rabbit, diluted 1:2000 in 3:1 TBS-T and NAP-blocker. Incubated at RT for 2 hours. Wash 30 minutes with TBS-T Attempted second time using 5:1 dilution of TBS-T and NAP-blocker. Wash 30 minutes with TBS-T 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? I have changed the amount of blocking reagent in the Primary, secondary, and Streptavadin solutions from 3:1 TBS-T/NAP-Blocker to 5:1 TBS-T/NAP-Blocker with no different results.
Asked on Jun 07 2006
Thank you for your enquiry. I am sorry to hear that you have been having difficulties with this antibody. I have read your technical questionnaire and I appreciate the additional steps that you have performed in order to try and get this antibody to work. I am certainly prepared to offer you a credit note against this purchase provided that the antibody was purchased within the past 90 days. If this is the case please email me details of the order including the order number and date of purchase. I look forward to hearing from you.
Answered on Jun 08 2006