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Loss of protein upon concentration


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#1 guyava

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Posted 20 January 2011 - 09:53 PM

I'm purifying a 110kD protein expressed in mammalian cells, using a FLAG column. The elution buffer is 1ml Glycine pH3.5 per fraction, eluted into 50uL of 10XTBS (i.e. 0.5M Tris pH7.4, 1.5M NaCl) to neutralize the acidic eluant. Before concentrating the protein, it is ~0.3mg/ml. However, since I need at least 1mg/ml, I am concentrating using Vivaspin microconcentrators, with a cutoff less than half the MW of my protein - i.e. 50kD. I spin for 1 minute and then mix the solution several times to avoid absorbance to the membrane. Although the volume decreases by 1:3 - 1:4, I seem to lose a lot of my protein, as the concentration after using Vivaspin is ~0.5mg/ml. How can I overcome this and not lose my protein? Is there any additive I should add to my buffer (preferrably not a detergent, as this will interfere with subsequent assays)?

Thanks!

#2 mdfenko

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Posted 21 January 2011 - 01:40 PM

you can try increasing the salt concentration to reduce non-specific binding to the membrane. you could double your neutralizing solution.

you may want to try a membrane with a lower mw cutoff (i would use 10 kDa or less). if your protein is not globular then it may be passing through the pores of the membrane.
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#3 DeeDee

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Posted 22 January 2011 - 01:25 AM

You could use glycerol in your buffer to 10%... I dont know how this works,but I could achieve good protein recovery while using glycerol containing buffers as opposed to the no glycerol ones.

#4 guyava

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Posted 22 January 2011 - 08:21 PM

Thanks all - I'll try using Glycerol (~5%) and lower the cutoff. I'll keep you posted.

Thanks!

#5 SebS

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Posted 09 February 2011 - 02:05 AM

not sure if you still need help with this but I have found that my protein stuck to the concentrator membrane. Other than that I'm facing the exact same problem. Immunoaffinity purified protein elutes nicely to 0.4mg/ml. Concentrated it once to 0.92mg/ml (500ul) and injected on a S75 column, where I had great separation and nice peaks. Collected peak containing fractions, nano-dropped them (very little protein left!! Like 0.12mg total $ 0.08mg/ml in 1.5ml). Concentrated the protein to 500ul and now left with 0.1mg/ml, so around 50ug left.

No idea how that can happen! The protein concentrated fine before, but I lost it after concentrating for a second time (using the same concentrator). Note, no precipitates formed and I'm using 10% glycerol in my buffer as well. Even stranger is the fact that what went into the gel fil definitely didn't come out into my collection tubes...
Any comments would be appreciated!

Edited by SebS, 09 February 2011 - 02:07 AM.





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