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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-

luciana on Apr 9 2010, 03:12 PM said:

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


Do you use SDS-PAGE or native PAGE? In the case of SDS-PAGE: To avoid aggregation do not boil samples at 100C but use 65C or even 30C with appropriate longer incubation

-Inmost sun-

Hi, thanks for your answer, I use SDS-PAGE, and of course, i boil at 100C!!!
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



Do you use SDS-PAGE or native PAGE? In the case of SDS-PAGE: To avoid aggregation do not boil samples at 100C but use 65C or even 30C with appropriate longer incubation

-luciana-

Inmost sun on Apr 14 2010, 08:42 AM said:

luciana on Apr 9 2010, 03:12 PM said:

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


Do you use SDS-PAGE or native PAGE? In the case of SDS-PAGE: To avoid aggregation do not boil samples at 100C but use 65C or even 30C with appropriate longer incubation


larger proteins tend to aggregate if heated harshly; so incubation at 30C may work better in your case; in such case we use a "power Laemmli" sample buffer which means containing additional protease inhibitors

-Inmost sun-