FLAG, Sigma-Aldrich, F3165

M2 anti-FLAG tag (murine IgG1)
Product # F3165 from Sigma-Aldrich

This is the standard antibody which I have used for a variety of applications in detecting several recombinant FLAG-tagged proteins. Very reliable.

ELISA: used at 0.5 ug/mL in 2% BSA PBS, followed by detection with a GAM-HRP

Western blot: used at 0.5 ug/mL in 2% milk powder – PBS, then detected with GAM-HRP

FACS: used at 5 ug/mL in 1% FBS PBS, then detected with GAM-PE

Western Blot detection of transfected HEK293T transient expressing FLAG tagget Cas9 protein. Only Faint unspecific signal on 30µg/lane (1:5000 HRP pRb)

Dilution 1:2000

This antibody works fine for the detection of a C-terminal FLAG-tagged protein in rat primary oligodendrocytes.

Dilution 1:100

I have used this antibody against a FLAGtag in the c-terminal of a receptor protein, with some success. The antibody needs to be fresh, as the stock in the fridge did not work. A fresh stock from -20C worked great, with comparable signal strength to an antibody against the actual receptor protein. The M2 ab is Calcium independent and bind FLAG tag anywhere in the protein.

Dilut 1:1000.

This antibody dimerises/polymerises exogenous FLAG-tagged sFasL in cell cultures, which dimerises/polymerises FAS in jurkat cells. I used 1 ug/mL in cell culture medium for 5 hours at 37 C.

Works for the detection of FLAG-tagged proteins expressed in HEK293 cells by immunofluorescense.
Dilution 1:500

Works OK for IP