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.
On October 29, 2017 Heidi Gytz Olesen, McGill University, Canada wrote:
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.