This is my first time doing this and I would like some input:
-MY first step would be to run a gradient of AS% saturation in crude lysate to observe the min and max req for protein precipitation.
-Say I observe 20% (w/v) as producing an optimal band density on SDS PAGE. How would I decide how much buffer to resuspend the pellet in? Is it a matter of simply how concentrated I want my protein (i.e. add less buffer for higher conc.?)
-After resuspending my pellet and dialyzing out AS, I would concentrate via a Vivaspin unit but there's always brown residue clogging the filter. Is this the result of inadequate mixing in the step prior?
-After putting the concentrated product into SEC on AKTA I would find a peak corresponding to aggregated protein right next to my target. What are some ways I can eliminate this aggregate protein peak?
Thanks in advance for your help.
Add solid ammonium sulfate gradually, with magnetic stirring, to reach each desired percentage of saturation, until the salt is dissolved. After allowing to sit for an hour in the cold, centrifuge the sample at 10,000 g for 10 min. Save the pellet for analysis. Add more ammonium sulfate to the supernatant to reach the next step, as before. Continue this procedure until you have tested each desired concentration (e.g. 10, 20, 30, 40, 50, 60, 70, 80% of saturation). Consult a published table for the amount of solid ammonium sulfate needed to reach each desired concentration.
If you have enough sample, you can speed up this procedure by making simultaneous samples for each ammonium sulfate concentration instead of sequential ones.
Dissolve each pellet in a small volume of buffer and dialyze overnight in the cold against a buffer to remove the ammonium sulfate (because it will mess up the SDS-PAGE). Measure the protein concentration of each dialyzed sample and use that information to prepare samples for SDS-PAGE with equal amounts of protein. Use SDS-PAGE to identify the ammonium sulfate concentration at which the desired protein precipitates most selectively.
Another potential goal of this procedure is to identify the lowest concentration of ammonium sulfate needed to precipitate most of the protein.
With this information, you can design the fractionation protocol. Use a lower concentration to precipitate other proteins. Centrifuge the sample to remove those. Then add just enough solid ammonium sulfate to the supernatant to precipitate the desired protein. Centrifuge again to collect the pellet.
SDS-PAGE is not the only method you could use to test for the protein of interest. You could use a functional assay or an immunological one (Western or ELISA). These methods are most suitable when the level of protein expression is not high enough to make it an obvious band on SDS-PAGE.
Adam B Shapiro Thank you for the thorough explanation. How would you usually decide how much buffer to resuspend before dialyzing? Is it a matter of wanting concentrated or less concentrated sample? Do you recommend concentrating with a spin column if after dialysis, I aim to perform SEC?
The volume for resuspension depends on the size of the pellet. There is always some ammonium sulfate solution remaining with the pellet, and the protein may have a limit to its solubility. So, if the volume is too small, the pellet will not fully dissolve. You can add more buffer if needed.
Another consideration is the type of tubing or dialysis cassettes you have available. If you have wide tubing, you need to use a larger sample volume than if you have narrower tubing.
A faster alternative to dialysis is to use a desalting gel filtration column, either by gravity or centrifugation. The sample volume requirement will be dictated by the column requirements, in that case.
You can use a centrifugal ultrafiltration device to concentrate the sample after dialysis.
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