Sigma Aldrich
ANTI-FLAG® antibody produced in rabbit
F7425-.2MG
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Our lab uses Anti-FLAG® M2 Magnetic Beads (produced in mouse, Sigma) for protein interaction co-immunoprecipitations. Invariably, some of the mouse antibody carries through with the eluted sample, especially as we generally use protein sample buffer for elution. This causes background in the western blot when detecting the FLAG epitope protein using a mouse-directed secondary antibody. A great way to avoid this problem is to use a FLAG antibody raised in rabbit, so that a rabbit-directed secondary may be used. The dramatically reduces background in a Western blot.
Western blot
Protein samples from a co-immunoprecipitation
1:5000 in 5% skim milk in TBST, 4 degrees C, 16 hours
5% skim milk powder in TBST
Goat anti-rabbit in 5% skim milk powder in TBST, 1 hour room temperature
NA
Enhanced Chemi-luminescence, exposure to film
The banding pattern should be clean, and at the correct size of the FLAG epitope containing protein.
Antibody may be re-used if stored at 4 degrees C with sodium azide.
Very clear Western blots with no antibody background from M2 beads.
Expensive.
Great for anti-FLAG M2 agarose bead immunoprecipitations.