dimerization or aggregation of my protein! - (Apr/09/2010 )
Hi all,
I made western blot of my membrane protein of interest. MW:120kDa. using a new primary antibody, since the old one i used in repeated westerns did not work!!
Fortunantly, I could see my protein in the right molecular weight. However, I also see a band at the top of the filter (up to 250kda)!
Im not familiar with the terms of dimer, monomer... so, I dont know the cause of this band!!
if the protein was aggregated in the sample, ( i used loading buffer containing DTT).
all the suggestions are welcome
thanks
luciana on Apr 9 2010, 03:12 PM said:
I made western blot of my membrane protein of interest. MW:120kDa. using a new primary antibody, since the old one i used in repeated westerns did not work!!
Fortunantly, I could see my protein in the right molecular weight. However, I also see a band at the top of the filter (up to 250kda)!
Im not familiar with the terms of dimer, monomer... so, I dont know the cause of this band!!
if the protein was aggregated in the sample, ( i used loading buffer containing DTT).
all the suggestions are welcome
thanks
Do you use SDS-PAGE or native PAGE? In the case of SDS-PAGE: To avoid aggregation do not boil samples at 100°C but use 65°C or even 30°C with appropriate longer incubation
Hi, thanks for your answer, I use SDS-PAGE, and of course, i boil at 100°C!!!
so, tomorrow, i will not boil, and leave in laemly buffer at RT for 30min, what do you think??
thanks alot
I made western blot of my membrane protein of interest. MW:120kDa. using a new primary antibody, since the old one i used in repeated westerns did not work!!
Fortunantly, I could see my protein in the right molecular weight. However, I also see a band at the top of the filter (up to 250kda)!
Im not familiar with the terms of dimer, monomer... so, I dont know the cause of this band!!
if the protein was aggregated in the sample, ( i used loading buffer containing DTT).
all the suggestions are welcome
thanks