How to choose the ready gel for western blotting - (Oct/11/2007 )
hi guys,
I'm going to detect a kind of protein 140kd size by western blotting. I found there are different kinds of ready gel in BIO-RAD. One is single concentration of Tris-HCL(7.5%) and another is 4-20% Tris-Hcl. I'm not sure which one I should choose and what's the difference between them?
By the way, how many proteins normally should I put in each well when do the SDS-PAGE?
Thanks in advance.
I'm going to detect a kind of protein 140kd size by western blotting. I found there are different kinds of ready gel in BIO-RAD. One is single concentration of Tris-HCL(7.5%) and another is 4-20% Tris-Hcl. I'm not sure which one I should choose and what's the difference between them?
By the way, how many proteins normally should I put in each well when do the SDS-PAGE?
Thanks in advance.
it is no matter of BIORAD but a general question:
7.5%: you separate only proteins ~>100 kDa, bands are broad
4-20% separation of nearly all proteins, better separation for proteins ~>100 kDa, sharper but a lot more bands
I would take a gradient gel
As Bearer has already explained, I would also prefer to go with the gradient 4-20% gel.
Yes, as the both before said, take the gradient gel for better resolution and entry in the gelmatrix of big proteins.
As to the amount of loaded protein, it depends a bit on the expression level of your protein in the cell. Normally, amounts between 25-30 µg should be o.k., but if it is really only weakly expressed you can go up to 50 µg. Depends also on the size of the gel and the number of comp teeth, the amount may is limited to a certain volume (20 µl für 15 teeth, 40-50 µl in 10 teeth), so perhaps you may have to concentrate your sample, if it is too diluted.