Is it possible to obtain readable results using a loading control conjugated antibody (e.g., Rb anti-Gapdh 680) along with a target protein unconjugated antibody plus secondary, if the target protein antibody is also raised in rabbit (e.g., Rb anti-Trpv1 + Gt anti-Rb 800)?
Ideally, the conjugated antibody shouldn't have any free sites for the secondary to bind, but I'm concerned that there will be some cross-reactivity rendering the blot uninterpretable. We are hoping to be able to stick with proven antibodies rather than seeking out other species.
Apart from that, is it advisable to pair one conjugated and one unconjugated antibody in a multiplex blot in general (i.e. stain for conjugated antibody and primary concurrently, then wash, then stain for secondary)? Thanks for any advice!
This is not an answer to your question, only a comment. It's an interesting idea. By employing both single- and double-amplified signals in a single assay, you will compromise quantitation (which is sketchy on WB, anyway), but the qualitative result should yield usable data, as long as your targets are well-separated on the blot. Good luck!
You certainly can do this using 1o Ab from different species, developed with 2o Ab bearing different labels for each species.
I have successfully multiplexed WB by developing for one Ag, then stripping the blot (with Gly-HCl) and developing the 2d Ag. I also have done this by using scissors to split the blot into strips, and separately developing the strips for two target Ag. To line them up for comparison, you need to be careful to cool the electrophoresis to minimize "smiling."
Different color bands on a single WB lane for your multiple target proteins makes for a striking figure, although any time you employ multiple colors on a figure you should consider 508 compliance.
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