Editing PEI Precipitation (section)

=== Removal of PEI from Eluted Protein<br> <br> ===

<br> When protein is eluted from the PEI pellet, PEI is also released into solution. If the eluate is dialyzed or diluted to lower ionic strength, the free protein and PEI will again interact and precipitate. Therefore, one must remove the free PEI. This is most commonly done by adding enough ammonium sulfate to the eluate to precipitate the protein. At this high ionic strength (often 40-60% saturated ammonium sulfate), PEI will not bind to protein and will remain in the supernatant after the ammonium sulfate precipitate is pelleted. This procedure removes the vast majority of the PEI and is sufficient for most proteins. If a protein is especially sensitive to residual traces of PEI, then a further step is required. The easiest method is to thoroughly resuspend the ammonium sulfate pellet in 40-60% saturated ammonium sulfate (sufficient to keep the protein insoluble) and recentrifuge. This way, any PEI physically trapped in the pellet will be removed. Another method to remove PEI involves passing the eluate, or the dissolved ammonium sulfate precipitate, through a phosphocellulose column at 1 M NaCI. The protein will not bind to the column at this high ionic strength, but the PEI binds very tightly and is completely removed. <br>

must remove the free PEI. This is most commonly done by adding enough ammonium sulfate to the eluate to precipitate the protein. At this high ionic strength (often 40-60% saturated ammonium sulfate), PEI will not bind to protein and will remain in the supernatant after the ammonium sulfate precipitate is pelleted. This procedure removes the vast majority of the PEI and is sufficient for most proteins. If a protein is especially sensitive to residual traces of PEI, then a further step is required. The easiest method is to thoroughly resuspend the ammonium sulfate pellet in 40-60% saturated ammonium sulfate (sufficient to keep the protein insoluble) and recentrifuge. This way, any PEI physically trapped in the pellet will be removed. Another method to remove PEI involves passing the eluate, or the dissolved ammonium sulfate precipitate, through a phosphocellulose col- umn at 1 M NaCI. The protein will not bind to the column at this high ionic strength, but the PEI binds very tightly and is completely removed. <br><br>

[[Category:Protocols]]

<br>