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BATCH NUMBER 77744 ORDER NUMBER PR340939 DESCRIPTION OF THE PROBLEM WB on human cell lysates expressing a mammalian expression vector encoding human TRF1 tagged with a flag epitope tag. The immunodetection of flag-TRF1 protein could be seen on immunoblots using anti-flag antibody (Sigma) but no specific band at the expected molecular weigth was observed when the menbrane was hybridised with Ab14397. SAMPLE human fibroblasts cell lysates stably expressing a mammalian expression vector encoding human TRF1 tagged with a flag epitope tag. PRIMARY ANTIBODY Ab14397 dilution 1/250 in (TBS-T) 3 hr at room temperature and O/N at 4?C membrane washed with TBS-T 0,05% 3 times for 5 min at room temperature DETECTION METHOD enhanced chemiluminescence was used (Cat NEL105 ; Amersham) product currently used in the lab POSITIVE AND NEGATIVE CONTROLS USED A tagged TRF1 protein was overexpressed in the test ANTIBODY STORAGE CONDITIONS Antibody arrived in solution and was used immediately after delivery on Friday the 10th of February. Now, the antibody is aliquoted and stored at -20?C SAMPLE PREPARATION lysis buffer used (condition optimized for the detection of another telomeric protein TRF2) 50 mM tris-HCl, pH 7,4 ; 1% NP40 ; 0,25% sodium deoxycholate; 150 mM NaCl ; 1 mM EGTA ; 1 mM PMSF ; 1microgram/ml of leupeptin, aprotinin ; pepstatin; 1 mM Na3Vo4 ; 1 mM NaF boiled samples AMOUNT OF PROTEIN LOADED 60 microgram of total cell lysates ELECTROPHORESIS/GEL CONDITIONS 8% polyacrylamide gel and reducing condition TRANSFER AND BLOCKING CONDITIONS PVDF membrane was used blocking inTBS containing 5% nonfat dry milk and 0,1% Tween (TBS-T) 20 for 45 min at room temperature SECONDARY ANTIBODY sheep anti-mouse HRP conjugated IgG ( [a competitor], product currently used in the lab) dilution 1/5000 in (TBS-T), 1h at room membrane washed with TBS-T 0,05% 3 times for 5 min at room temperature HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1
Asked on Feb 15 2006
Many thanks for taking the time to provide all your protocol information, it is very useful to understand your problem and I'm sorry to hear you are experiencing unsatisfactory results with ab14397. I think your protocol is very good and the fact that your flag tag is detected is very positive; however, I am wondering if you are not expressing the whole sequence of the protein and as it is a monoclonal antibody it will only recognize one single epitope. I would therefore recommend to run a positive control along your samples, we recommend HeLa nuclear lysate. I was able to find out that the antibody was used at 1:100-1:200 so your choice of 1:250 is only a little above that dilution and should be ok. Please let me know your thoughts on the matter of the protein sequence, I look forward to hearing from you soon,
Answered on Feb 16 2006