Questions for regarding sucrose gradients - (Feb/14/2011 )
So, my work in the lab has focused on identifying interacting proteins for a particular protein-of-interest my lab studies. I've been doing this through immunoprecipitation/Mass spec, and have identified a rather large protein complex (~1-1.5 MDa) that interacts with my protein. I am interested to see if I can detect this complex co-migrating with my protein via sucrose gradients, and just had some general questions about optimizing my gradients:
1. How important is it to collect fractions from the bottom up? Some other members in my lab have collected fractions from the top down (using very careful pipetting), and this seems to work okay, but most protocols I've found suggest somehow punching a hole in the bottom of the tube and collecting fractions that way. Does anyone know how much of a difference this makes?
2. Does the step size of the sucrose gradient affect the quality of the gradient? i.e, Does a gradient that goes 40%-30%-20% work as well as a 40%-35%-30%-25%-20% gradient of the same final volume?
3. What is the best way to get a general idea of the size of the complex in a given fraction? I've tried simply running my samples in duplicate and boiling one of them to get denatured protein, so I can maybe see the general fraction at which they run when not in complex, but I don't know if that's the best way.
Any help is appreciated. Thanks.
Velox on Mon Feb 14 21:24:45 2011 said:
1. How important is it to collect fractions from the bottom up? Some other members in my lab have collected fractions from the top down (using very careful pipetting), and this seems to work okay, but most protocols I've found suggest somehow punching a hole in the bottom of the tube and collecting fractions that way. Does anyone know how much of a difference this makes?
if you are careful and keep track of the fractions then it makes no difference. there is (was?) apparatus to pump dense solution into the bottom of the tube so that the gradient would be fractionated from the top.
more steps give a finer gradient which may give finer separations and a better idea of density of the protein.
you can apply some of the fraction to sds-page or native page to look at the protein content of the fractions.
Velox on Mon Feb 14 21:24:45 2011 said:
2. Does the step size of the sucrose gradient affect the quality of the gradient? i.e, Does a gradient that goes 40%-30%-20% work as well as a 40%-35%-30%-25%-20% gradient of the same final volume?
Hi,
I'm not an expert, but there is a commercially available device to prepare sucrose gradients. I use it to prepare gradients to purify vaccinia virions.
You can prepare a gradient ranging from 40 to 20% that, I guess, will give you a better separation than gradients you cited above.
here is a link for one of those gradient prep devices. they're called gradient mixers
http://cell-lysis.com