Question 1: What is the best RhoA antibody available?
Answer 1: There are multiple options when choosing an antibody to detect small GTPases. Several factors must be considered: specificity, sensitivity, format compatibility, species cross-reactivity and consistency of signal. Cytoskeleton’s RhoA mouse monoclonal antibody (Cat. # ARH03) scores high marks on all of these parameters. It is specific to RhoA, meaning not only will it not detect Rac or Cdc42 proteins, but it is isotype specific as well. It will not detect RhoB or RhoC. It is also very sensitive and works well for both western blotting and immunochemistry. Of course there are other companies (e.g. Cell Signal Technologies) that also make good antibodies that are comparable to ours. There have been reports of lot to lot variability with some supplier’s RhoA antibodies. Cytoskeleton’s antibodies do not have batch to batch variability due to our stringent quality control process.
Answer 2: There are multiple options when choosing an antibody to detect small GTPases. Several factors must be considered: specificity, sensitivity, format compatibility, species cross-reactivity and consistency of signal. We have performed direct comparisons between our Rac1 specific mouse monoclonal antibody (Cat. # ARC03) and that of our competitors, and we have found that our Rac1 antibody is the only one that is truly specific for Rac1. Others detect either Cdc42, Rac2 or Rac3 proteins in addition to Rac1. Our Rac1 specific antibody works for both western blotting and immunochemistry.
Answer 3: All dilutions are noted in the datasheet. The actual dilution of antibody depends on the cell/tissue type and experimental format being used. For western blotting, we have tested and recommend using the antibody at a concentration of 500 ng/ml (1:1000 dilution). At this dilution, we detected tubulin in extracts of Drosophila S2 cells, Xenopus A6 cells, mouse Swiss 3T3 cells, rat NRK cells, human HeLa cells, and bovine brain. For immunofluorescence, we used the anti-tubulin antibody to detect tubulin in mouse Swiss 3T3 cells. The antibody was used at a concentration of 2.5 μg/ml (1:200 dilution) followed by incubation with a 1:500 dilution of anti-sheep rhodamine-conjugated secondary antibody. This information is found usually in the figure legends or within the “Product Uses” section of our antibody datasheets.