hi,
Iam working with a halotag attached to my protein expressed in Hela cells. I was trying to pull down the halotag with magne beads and got nothing in the western. I tried increasing the binding time to three hrs and nothing worked. So took the cell lysis ,did the western and probed with halotag antibody, but was not able to detect the protein. I tried the lysis with and without the detergents. Iam able to see the halotag and my protein after addition of TMR in the microscope, but somehow Iam not able to see it in the western, Checked with the company for the antibody and they say all good. Could anyone help me out with this.
thanks
jaya
Have you used protease inhibitors in your lysis buffer? If not, that might be a problem.
As the manual says, you can also use TMR ligand for western blotting. Have tried that? I feel you are losing your tag during lysis. Check the lysate with TMR in western. With this, you will know whether you are losing the tag during lysis or not. I read the following manual, which is very interesting. I hope you have the same. If not please find the attached. Goodluck
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