protein lost on membrane of centricon!!

From: rashmi panigrahi
Date: 28 January 2012 15:54


Hi all,
I tried to concentrate my protein using vivaspin 20  10,000 MWCO PES.
The protein was in 50mM Hepes pH 7.5, 500mM KCl  and 10% glycerol.
I lost about 90% of my protein on the membrane of the centricon.

Please  suggest some way of concentrating this protein.
Will concentrating using peg 20K be a good alternative??

regards

rashmi

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From: Cale Dakwar

Hello Rashmi,

How large are your protein monomer units?  Are you expecting these units to have formed an oligomer?  As a general rule of thumb, you want your protein molecules to be no smaller than 3x the membrane MWCO.  Perhaps all you need is to try concentrating with a lower MWCO membrane, e.g. 3,000.

Alternatively, you could try:

- change membrane manufacturers (this sometimes does make a difference),

- pretreating your membrane (e.g. with BSA) to block all binding sites (but then you'll have to worry about BSA contamination of your sample),

- concentrating your protein in under nitrogen pressure (I forget right now what the device is called but I used to use it all the time),

- concentrating your protein in a speed vac to evaporate off some of the water,

- ammonium sulphate precipitation,

- or even TCA precipitation.

HTH,

C

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From: Cale Dakwar

P.S.  I haven't personally tried concentrating with PEG 20K but I suppose it could work.

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From: Bosch, Juergen

Are you close to the theoretical isoelectric point of your protein ? Change pH of buffer
Jürgen

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From: Doug Ohlendorf

Rashmi, 

 

There are other membrane materials which might bind less. We have also found that there seems to be binding sometimes to the plastic itself. So a different manufacturer can help.

 

Also, always use same centricon for same protein. Sometimes you take a big hit on a centricon's first use and then it is fine for subsequent uses. If the protein prep is not terribly difficult, you could try that centricon a second time.

 

We have used PEG 20 K to pull water out. If you do that you must watch it carefully and immerse the dialysis tubing in final buffer when you are near final concentration. If you just remove the PEG without washing it will continue to concentrate.

 

Doug Ohlendorf

 

 

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From: Bosch, Juergen

Another option I didn't mention is presoak your centricons in 30% glycerol over night prior to usage.

Jürgen

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From: AARON HERNANDEZ

Rashmi,

 

I had a similar problem when I used an amicon to concentrate my protein, your buffer composition indicates me that a lot of salt concentration and glicerol makes your protein soluble, I had a similar buffer with a lot of saltt and 15% glicerol, and I simply reason that my protein it is actually soluble but Temperature was also critical so I decided to take the centrifuge inside of a refrigerator and I centrifuge at 10°C or less and my protein survive the process of a possible thermal shock that the protein is induced, sometimes something simple works fine and it did for me.

 

hope this help you.

 

aaron hernandez

Universidad nacional autónoma de Mexico UNAM