Question (14105) | Anti-RASSF1a antibody [3F3] (ab23950)

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Question

BATCH NUMBER 132644 ORDER NUMBER -- NOT SPECIFIED -- DESCRIPTION OF THE PROBLEM low intensity of bands, many unspecific bands SAMPLE Total cell extract from human melanoma cell line IGR1; DETECTION METHOD Detection with BciP/NBT POSITIVE AND NEGATIVE CONTROLS USED Positive control: human fibroblast cell line HF53 (RASSF1a expressed); Negative control: human lung cancer cell line A549 (RASSF1a non expressed). SAMPLE PREPARATION RIPA-Buffer with ?complete? protease inhibitor coctail tablets (1complete tablete diluted in 50 ml RIPA-Buffer); the cells were prepared with 21G hypodermic needle. AMOUNT OF PROTEIN LOADED 10?g of total protein from each sample was loaded. TRANSFER AND BLOCKING CONDITIONS Transfer in NaH2PO4-Blotting-Buffer (2% 1M NaH2PO4; 1,4% 0,5M NaH2PO4); 1h; 350 Ampere; icecold. 2h Blocking with 10 ml TBS-Tween (0,1% Tween, 5% nonfat dried milk powder). SECONDARY ANTIBODY Anti-mouse IgG (goat) (competitor); Concentration 1,4mg/ml; 1/2000 diluted in TBS-Tween; Incubation time 2h. HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 1 DO YOU OBTAIN THE SAME RESULTS EVERY TIME? No ADDITIONAL NOTES Please send a detailed protocol for Western-Blot with RASSF1a mouse monoclonal antibody (ab23950). The Blot-Picture is attached to this e-mail.

Answer

Thank you for your patience and I'm sorry to hear that your customer is experiencing difficulty with this antibody. Our source for ab23950 was able to provide the following details regarding its characterization in Western blotting. The extracts of HeLa cells, mouse brain, mouse liver and mouse kidney tissues (each 20 ug) were resolved by SDS-PAGE, transferred to PVDF membrane and probed with anti-human RASSF1A (1:1000). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP and a ECL detection system. 293 cell transfected with the HA-RASSF1a gene was also used as positive control. Your customer didn't mention what dilution they tried, but I would suggest starting with 1:1000 with incubation overnight at 4C. Also, I would suggest loading 20-30 ug protein lysate (you customer mentioned 10 ug), and make sure to run a secondary-only control (omit the primary antibody) to ensure that the secondary antibody is not binding non-specifically. I hope this helps. If you need additional assistance, please contact us again.

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