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BATCH NUMBER 66531 ORDER NUMBER see end DESCRIPTION OF THE PROBLEM No signal at all, and the background is very clean (even after 30 minutes of exposure) SAMPLE 12 Human tumor cell lines including breast carcinoma (as positive control since the expression of p8 in breast cancer has been reported), prostate cancer, fibrosarcoma, cervix carcinoma and colorectal carcinoma. PRIMARY ANTIBODY abcam/rabbit anti-human/1:500/1 hour, 5 minutes 3 times in TBS-T SECONDARY ANTIBODY Oncogene/Goat anti-rabbit/1:3000/45 minutes, 5 minutes 3 times in TBS-T, then 5 minutes once in TBS DETECTION METHOD ECL, from Santa Cruz POSITIVE AND NEGATIVE CONTROLS USED Positive control for p8: 3 breast tumor cell line whole cell lysates ANTIBODY STORAGE CONDITIONS Performed one hybridization right after I received the antibody, i.e. before the aliquots and frozen. SAMPLE PREPARATION RIPA buffer with PMSF, Aprotinin and sodium orthovanadate. Samples were boiled for 5 minutes. AMOUNT OF PROTEIN LOADED approximately 30-50 ug/lane ELECTROPHORESIS/GEL CONDITIONS 15% SDS-PAGE gel TRANSFER AND BLOCKING CONDITIONS 10x Blotting buffer: 1 L 30.3 g Trizma base (= 0.25 M) 144 g Glycine (= 1.92 M) pH should be 8.3; do not adjust. To make 2 L of 1x Blotting buffer: 400 ml Methanol 200 ml 10x Blotting buffer 1400 ml water transfered at 15V constant voltage overnight Blocking: Blotto A: 1x TBS, 5% milk, 0.05% Tween-20 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? increased the primary antibody concentration to 1:200 dilution ADDITIONAL NOTES the same blots were incubated with anti b-action primary antibody with same conditions, and positive signal were detected. the messenger RNA level are very high in some of the cell lines (the same as that of b-actin or even higher) according to my Real Time PCR results. my P.O. number does not fit in the field. It's UHN5-014546. The order was placed through Cedarlane.
Asked on Nov 24 2004
I'm sorry to hear you are having problem with ab6028. P8 is a nuclear protein, do you do nuclear extraction to have a concentrated sample to load onto your gels? I would like to suggest an overnight incubation of the primary antibody (trying 1:200 and 1:500) at 4C diluted in TBST (0.1% Tween). I would also recommend human pancreatic cancer tissue as a positive control. Please let me know if this helps and do not hesitate to contact us again if you still have problems,
Answered on Nov 25 2004