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Gel filtration chromatography


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4 replies to this topic

#1 neuron

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Posted 31 May 2011 - 02:25 AM

Hi,

I need to fractionate the crude protein lysate in small-small fractions to separate the proteins, and for that I have to do gel filtration chromatography. I have never done it and now have to do it :angry: .......I asked people around, everybody does it for purification of single protein, nobody has done it on complex lysate. Has anyone done that??....if yes, please help me.....


Thanks

#2 nightingale

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Posted 31 May 2011 - 09:06 AM

Hi,

I need to fractionate the crude protein lysate in small-small fractions to separate the proteins, and for that I have to do gel filtration chromatography. I have never done it and now have to do it :angry: .......I asked people around, everybody does it for purification of single protein, nobody has done it on complex lysate. Has anyone done that??....if yes, please help me.....


Thanks


hello amj2 :)
i remember we did it, but very long ago !
here is a document i found : Attached File  GE_gelfiltration.pdf   1.77MB   205 downloads
hope helpful :)

" The more you learn, the more you realize how little you know ... "

#3 Luria Bertani

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Posted 31 May 2011 - 07:27 PM

Hi there,

I'm a bit surprised that what sounds like a raw lysate is going onto a Gel filtration column.

Usually there would be a column that removes most of the non-interesting proteins, such as Ion-exchange or Hydrophobic interaction chromatography
and then there would be a gel filtration as a polishing step.

If you load raw extract on to a gel filtration column, the end product will probably have a lot of other proteins that you don't want.

Cheers

LB

#4 neuron

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Posted 31 May 2011 - 11:04 PM

Thanks Nightingale and LB!!!.... Nightingalethe the document is really helpful :)
LB....You are absolutely right...actually we don't see our protein of interest on the gel whereas we see the band on the blot. its a small protein when i load concentrated lysate on the gel i see the streak...so the aim is to fractionate the lysate and then load on the gel ...when i was searching on the web for this, I came across a very nice intrument called gelfree8100 fractionation system...but unfortunately we don't have it and can't wait for it to come...so have to go for this gel filtration only...here is the link for gelfree8100-

gelfree8100

I hope gel filtration will help :(

#5 Luria Bertani

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Posted 01 June 2011 - 03:37 AM

What chromatography equipment do you have?

Usually if I wanted to fish out a protein from a soup of lysate (what kind of lysate?)
and there was no published protocol:


1. Find out the sequence and use this to calculate a predicted isoelectric point
2. Choose an ion exchange column (Q or SP Sepharose is useful) and a buffer that makes my
protein of interest charged and stick to the ion exchange column

3. run ion-exchange column, elute fraction off using NaCl

4. Figure out which fraction my protein is in using spectrophotometer or activity assay or SDS-PAGE Gel

5. Pool these fractions, concentrate them down (gel filtration needs concentrated sample)

6. Run gel filtration column




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