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I used the anti-Rab7 antibody to stain Vero and HeLa cells and did not observe any staining at all. Cells were seeded at 150,000 cells per well onto glass coverslips in a 6 well tissue culture plate and allowed to adhere overnight. Cells were washed with PBS 3x and fixed with 4% PFA for 10 min at room temp, and permeabilized with 0.5 % Triton X for 1 minute. Cells were washed with PBS 3x by 5 min each. Cells were blocked with 5% goat serum, 5% BSA prepared in PBS-Tween 0.5% for 1 hour at room temp, and incubated with primary rabbit-antiRab7 antibody (1:50 diluted in block) either for 1 hr at 37C, overnight at room temp, or overnight at 4C. Next day, cells were washed with PBS-Tween 0.5% and stained with secondary antibody (Fab fragment goat anti-H+L IgG Rabbit DyLight 649 at 1:200 dilution in block (1 hr at 37C) (Jackson immuno Research cat:111-497-003). Samples were washed 3x by 5 min with PBS and mounted with ProLong Gold containing DAPI and imaged using Leica TCS SP5 Confocal and multiphoton microscope. In addition to the 3 different primary antibody incubation conditions with 4% PFA, I also tried to fix cells with 1% glutaraldehyde with a sodium borohydride reduction and also did not observe any staining. I noticed that another customer also had a problem staining Vero cells with this antibody so I inquired about this with customer support and was assured that the problem was likely with the lot of antibody and that I should still go ahead with purchasing this antibody. However, it seems that it did not work for me either in both the human and monkey cells.
Asked on Oct 05 2012
Thank you for contacting us. I am sorry to hear that ab77993 is giving no signal on your samples. I would expect the protocol you are using to work well. If you have purchased the antibody in the past six months, I would be happy to offer you a free of charge replacement with one of our other RAB7 antibodies (such as ab50533) or a credit or refund. Please let me know your original order or PO number and how you would like to proceed and I will be happy to help you further.
Answered on Oct 05 2012