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Protein concentration with PEG
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WS
Apr 30, 2012, 7:50:03 AM4/30/12
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Dear Experts,
You are probably aware of the protein concentration method by
"dialyzing" against polyethyleneglycol. My question is regarding the
MW of PEG and the pore size of the tubing: Currently, I am using
PEG20000 (avg MW 15k-20k) and a dialysis tubing with a cutoff of 14kD
for globular proteins. In my experience, this is working very well
(and cheap), esp. for large volumes.
We just got some concerns now that some of the PEG could make it
across the membrane. Is there any need to be concerned? Which would
mean to switch to PEG35k or higher and/or tubing with a lower cut/off,
both increasing the expenses. As I understand PEG, the molecules will
get quite huge due to hydratation and PEG always has some low MW size
"contaminants" i.e. molecules shorter than desired.
Anything you can suggest?
Thanks!
Wo
You are probably aware of the protein concentration method by
"dialyzing" against polyethyleneglycol. My question is regarding the
MW of PEG and the pore size of the tubing: Currently, I am using
PEG20000 (avg MW 15k-20k) and a dialysis tubing with a cutoff of 14kD
for globular proteins. In my experience, this is working very well
(and cheap), esp. for large volumes.
We just got some concerns now that some of the PEG could make it
across the membrane. Is there any need to be concerned? Which would
mean to switch to PEG35k or higher and/or tubing with a lower cut/off,
both increasing the expenses. As I understand PEG, the molecules will
get quite huge due to hydratation and PEG always has some low MW size
"contaminants" i.e. molecules shorter than desired.
Anything you can suggest?
Thanks!
Wo
AllisonH
May 1, 2012, 11:13:32 AM5/1/12
to
How about Ficoll? The powder can be used just like PEG.
Allison
Allison
Message has been deleted
Dr Engelbert Buxbaum
May 19, 2012, 12:23:07 PM5/19/12
to
In article <jnphnv$npj$2...@news.albasani.net>, d...@noemail.thankstospam.net
says...
> get something into your protein. The same idea but much cleaner is to use
> dried polyacrylamide gel that swells a lot. That's what we do with proteins
> that cannot be concentrated by standard ultrafiltration. Buy this and be
> happy:
>
> http://www.spectrumlabs.com/dialysis/Absorbant.html
This of course has the disadvantage to cost some $20 per 5 ml sample.
There are two things to consider here: low molecular PEG that can cross
the membrane and other contaminants. The former are probably relatively
benign, especially when further purification steps follow. PEG actually
stabilizes protein conformation, that is why it is used in protein
crystallization.
Other contaminants are a different story, in particular peroxides. It
might be worthwhile to test each batch for those and to keep the PEG
dark, cool and dry.
That said, I have used the procedure many times, and never had any
problems. However, that was with 20 kDa PEG and a 6 kDa membrane rather
than 14 kDa as described by the OP.
says...
> >You are probably aware of the protein concentration method by
> >"dialyzing" against polyethyleneglycol.
> >
> >"dialyzing" against polyethyleneglycol.
> >
> >We just got some concerns now that some of the PEG could make it
> >across the membrane.
>
> I'd advise you against using PEG. All PEGs are "dirty" and you will inevitably
> >across the membrane.
>
> get something into your protein. The same idea but much cleaner is to use
> dried polyacrylamide gel that swells a lot. That's what we do with proteins
> that cannot be concentrated by standard ultrafiltration. Buy this and be
> happy:
>
> http://www.spectrumlabs.com/dialysis/Absorbant.html
This of course has the disadvantage to cost some $20 per 5 ml sample.
There are two things to consider here: low molecular PEG that can cross
the membrane and other contaminants. The former are probably relatively
benign, especially when further purification steps follow. PEG actually
stabilizes protein conformation, that is why it is used in protein
crystallization.
Other contaminants are a different story, in particular peroxides. It
might be worthwhile to test each batch for those and to keep the PEG
dark, cool and dry.
That said, I have used the procedure many times, and never had any
problems. However, that was with 20 kDa PEG and a 6 kDa membrane rather
than 14 kDa as described by the OP.
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