Aggregated proteins in SDS-PAGE - (Jul/10/2006 )
I need to see dimers of my protein in SDS-PAGE. If I used non-reducing conditions (no beta mercaptoethanol) the proteins formed big aggregates and did not migrate. What to do? Please, help me!
-Jaana-
QUOTE (Jaana @ Jul 10 2006, 02:31 PM)
I need to see dimers of my protein in SDS-PAGE. If I used non-reducing conditions (no beta mercaptoethanol) the proteins formed big aggregates and did not migrate. What to do? Please, help me!
I'm not sure but I would run a normal denaturating SDS-Page (including beta-meOH) but NOT cook samples before loading onto the gel.
I think dimers should persist this way...
-kylvalda-
Did you boil?
if you boil w/o beta mercaptoethanol, you will disrupt protein protein interactions, but not disulfide bounds, allowing to see dimers
-Missele-
QUOTE (Missele @ Jul 10 2006, 04:40 PM)
Did you boil?
if you boil w/o beta mercaptoethanol, you will disrupt protein protein interactions, but not disulfide bounds, allowing to see dimers
if you boil w/o beta mercaptoethanol, you will disrupt protein protein interactions, but not disulfide bounds, allowing to see dimers
Thank You for Your answer!
Yes, I did boil 5 min in 95 C. I read from the internet that sometimes hydrophobic proteins make more aggregates after boiling. Maybe I should try without boiling? Or should I use lower temperatures for denaturation? Like 60 or 70 degrees? Any opinions?
-Jaana-
I didn't know about hydrophobic proteins making aggregate after boiling.
Do you have enough samples? you could maybe try both.
I'm sorry, I have no idea.
-Missele-
QUOTE
Thank You for Your answer!
Yes, I did boil 5 min in 95 C. I read from the internet that sometimes hydrophobic proteins make more aggregates after boiling. Maybe I should try without boiling? Or should I use lower temperatures for denaturation? Like 60 or 70 degrees? Any opinions?
Yes, I did boil 5 min in 95 C. I read from the internet that sometimes hydrophobic proteins make more aggregates after boiling. Maybe I should try without boiling? Or should I use lower temperatures for denaturation? Like 60 or 70 degrees? Any opinions?
I've read that too - you can try 30 min 60 degrees...
Maybe you should do some tests on not so important samples like protein extraction from wild type tissue or cells....
-kylvalda-