His tag detection problem - (Oct/23/2005 )

I expressed a ~65 kd outermembrane protein using pET21b in BL21 (DE3) cells. The recombinant protein has a C-terminal his tag. Though I can see the expression of the induced protein in the SDS-PAGE, I cannot detect it in the western blot using anti His-tag antibody. I sent my clone for sequencing and the His-tag is in frame, so Iam really confused as to what is happening. Can somebody solve my problem?

I used the antibody from Sigma. I do use a positive control in the western blot which works. The positive control is also an outer membrane protein of similar size with a C-terminal His tag. I boil the outer membrane isolates for 5 minutes at 100 degree centigrade. The same conditions work for the positive control and not my protein.

Aarthi

QUOTE (aarthi @ Oct 24 2005, 06:38 AM)

I expressed a ~65 kd outermembrane protein using pET21b in BL21 (DE3) cells. The recombinant protein has a C-terminal his tag. Though I can see the expression of the induced protein in the SDS-PAGE, I cannot detect it in the western blot using anti His-tag antibody. I sent my clone for sequencing and the His-tag is in frame, so Iam really confused as to what is happening. Can somebody solve my problem?

Which antibody did you use ?
Do you have a positive control in your western blot ?
Did you extract your protein in native or denaturing conditions ?

Some of the anti His Tag that we generate in your lab are not recognizing every His tag recombinant protein !

Pesji