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
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3 replies to this topic
#2
Inmost sun
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Posted 14 April 2010 - 12:42 AM
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
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
#3
jasmina
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Posted 14 April 2010 - 03:12 AM
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[/quote]
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
[/quote]
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[/quote]
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
[/quote]
#4
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Posted 14 April 2010 - 03:36 AM
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
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
larger proteins tend to aggregate if heated harshly; so incubation at 30°C may work better in your case; in such case we use a "power Laemmli" sample buffer which means containing additional protease inhibitors
