DYKDDDDK tag, Genscript, A00187

THE DYKDDDDK (Flag) Tag, mouse, Genscript A00187

Dilutions:
1:1000 for immunoblotting
1:500 for IFC
IP: 1ug/1.5mg beads (Dynabeads® Protein G kit)
6.25ug/1.25mg beads (Co-IP kit, protein A Dynabeads)

short protocol:
For immunoblotting I dilute in 5% low fat milk powder in PBS-T and incubate overnight at 4°C.

Dynabeads kits (Invitrogen) were used for IP. Longer incubation of the antibody in the Ab binding and washing buffer of 1 h was used for optimal binding. Furthermore, mild washing conditions with low salt improved binding of the target protein.

For IFC the antibody was diluted in 3% BSA in PBS-T and incubated with the coverslip overnight at 4°C or alternatively, 1h at RT.

Review:
Performed very well overall. For the small hydrophobic proteins in our study, we found that it often worked better than the commonly used M2 antibody from Sigma.