concentrating low expression protein for western - will this work? (Feb/03/2008 )
Hi Guys,
Hoping to get some feedback on whether this will work or not.
I'm trying to detect a low expressing protein on a western blot (I have posted a few questions about this before). What I'm thinking of doing is an ultrafiltration to concentrate the protein, but at the same time I don't want to get a lot of higher molecular weight material because I fear that would overload the gel. The protein of interest is about 10 kDa. I thought that I could make plant cell lysates and pass them over a centricon filter (30 kDa). I would take the flow through from this, which should contain smaller molecular weight material (<30) and then pass this over a 5 or 10 kDa centricon filter. From this I would take the filtrate which should have retained and concentrated my protein and run the western.
My biggest concern is that there might be an issue with salt concentration. Also I've noticed when I run a normal western blot (with about 60 ug total cell lysate) that I see a large smear of green stuff (perhaps chlorophyll???) running around 20 kDa or so. I'm afraid that I might end up concentrating this as well and this would make the prep harder to work with.
I would appreciate any comments on this idea - or if you have a better suggestion, I'm all ears. 
Thanks,
smu
hi,
what about a gel filtration exp (Sephadex G-50)?
Sebastien
what about a gel filtration exp (Sephadex G-50)?
Sebastien
Thanks for the suggestion. When I proposed my method to my boss he actually says that he thinks gel filtration will work better too - for the simple reason that we don't know if my protein will be in a complex with other proteins and so it might not flow through the column. So I'm going to try that instead.
Hoping to get some feedback on whether this will work or not.
I'm trying to detect a low expressing protein on a western blot (I have posted a few questions about this before). What I'm thinking of doing is an ultrafiltration to concentrate the protein, but at the same time I don't want to get a lot of higher molecular weight material because I fear that would overload the gel. The protein of interest is about 10 kDa. I thought that I could make plant cell lysates and pass them over a centricon filter (30 kDa). I would take the flow through from this, which should contain smaller molecular weight material (<30) and then pass this over a 5 or 10 kDa centricon filter. From this I would take the filtrate which should have retained and concentrated my protein and run the western.
My biggest concern is that there might be an issue with salt concentration. Also I've noticed when I run a normal western blot (with about 60 ug total cell lysate) that I see a large smear of green stuff (perhaps chlorophyll???) running around 20 kDa or so. I'm afraid that I might end up concentrating this as well and this would make the prep harder to work with.
I would appreciate any comments on this idea - or if you have a better suggestion, I'm all ears.
Thanks,
smu
If you're not concerned about using the protein after concentrating, you could freeze-dry the whole volume, which will avoid the problem of where the protein has gone (filtrate/flowthrough). Then resuspend in a small volume for the gel step. You cold even do a bit of fractionation with the resuspended material to see if there are any associations with other proteins.