Editing PEI Precipitation (section)

== PEI precipitation of Protein ==

Optimization in Small Scale. Two-milliliter portions of extract are placed in 11 small centrifuge tubes and 5% (w/v) PEI stock solution is added to give final PEI concentra- tions of from 0 to 0.5% (w/v) in 0.05% (w/v) intervals. 2. Tubes are rapidly mixed after PEI addition, incubated for 5 min at 0°, mixed again, and centrifuged for 5 rain. 3. Supernatants are assayed for protein concentration and for the pres- ence of the enzyme of interest. This may be done by enzymatic assay, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, or by immunologic assay. total protein precipitated by PEI will depend on the pH (at higher pH, more proteins will carry a negative charge and be precipitated) and on the ionic strength (increasing ionic strength will weaken PEI-protein interactions). At low ionic strength (0-0.1 M NaCI), DNA and all acidic proteins will be precipitated. At higher ionic strength (0.1-1 M NaC1), only DNA and highly negatively charged proteins will precipitate. At greater than 1 M NaC1, only nucleic acids will precipitate. DNA -PEI precipitates dissolve at 1.4 to 1.6 M NaC1. T<br>



'''Strategy A''': Precipitate with PEI at high ionic strength to remove nucleic acid. Almost no proteins will bind to DNA or to PEI in 1 M NaCI. Therefore one can adjust the salt concentration of an extract to 1 M NaCI and titrate with PEI to achieve precipitation of nucleic acids, leaving the proteins in the supernatant. This titration can be conveniently followed by determining the UV absorption spectrum of the supernatants 9 and adding PEI until the supernatant shows an A280value greater than the A260, characteristic of protein solutions. While this strategy can be used, it is not common. One reason is that while nucleic acid is effectively removed, no purification from other proteins is achieved, unlike strategies B or C&nbsp; (below). Another problem is that if more PEI is added than is needed to precipitate the nucleic acids, excess PEI will be present in the supernatant with the proteins. When the supernatant is then diluted or dialyzed to<br>lower ionic strength, PEI-acidic protein complexes will precipitate. This excess PEI can be removed by ammonium sulfate precipitation as described below.<br><br>

'''Strategy B''': Precipitate with PEI at low to medium ionic strength to remove nucleic acids and some proteins, leaving enzyme of interest in supernatant. This is like strategy A but, because it is carried out at lower ionic strength, a substantial number of proteins will be precipitated. The enzyme of interest will remain in the supernatant and will not only be nucleic acid free but also will be free of the precipitated acidic proteins. This strategy works well for neutral or slightly basic DNA-binding proteins that do not bind to DNA under the ionic conditions of the PEI precipitation. <br>

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'''Strategy C''': Precipitate with PEI at lower ionic strength to precipitate nucleic acids and some proteins and then elute desired enzyme out of pellet with higher ionic strength. This strategy is by far the most commonly used. Generally one chooses a pH of 7.5-7.9 and an ionic strength of 0.1 to 0.2 M NaCI and then performs a PEI titration test to determine the minimal amount of PEI solution that causes complete precipitation of the enzyme of interest. The pellet, which contains nucleic acids and some proteins, is resuspended in a predetermined low ionic strength buffer to wash out any trapped proteins but not the desired enzyme. The washed pellet is then&nbsp; resuspended in a buffer of predetermined higher ionic strength to elute the enzyme, leaving the nucleic acids in the precipitate.&nbsp; The precise amounts of PEI added and the ionic strength of the wash and elution buffers are determined empirically as described below. The eluate contains the enzyme of interest, some other proteins, and a certain amount of PEI that was originally coprecipitated with the eluted proteins. The PEI is generally removed by ammonium sulfate precipitation of the protein, leaving the PEI in the ammonium sulfate supernatant. The ammonium sulfate pellet is dissolved in buffer and subsequent purification steps are performed.<br><br>

=== Preparation of stock<br> ===

100 ml&nbsp; of 50% (w/v) PEI is dissolved in 800 ml of deionized water. The solution is adjusted to pH 7.9 by adding concentrated HC1 (approximately 38-40 ml) with stirring. Dilute to 1000 ml with H<sub>2</sub>O, resulting in a clear, slightly bluish solution. This 5% (w/v) or 10% (v/v) stock solution is stored refrigerated and appears to be stable indefinitely.<br>

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=== Precipitation of DNA/RNA and/or protein by PEI.<br> ===

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'''Procedure '''<br>

1. 20 ml portions of extract are placed in 11 small centrifuge tubes and 5% (w/v) PEI stock solution is added to give final PEI concentra-tions of from 0 to 0.5% (w/v) in 0.05% (w/v) intervals. <br>

2. Tubes are rapidly mixed after PEI addition, incubated for 5 min at 0°, mixed again, and centrifuged for 5 rain. <br>

3. Add ni beads to bind target protein.<br>

4. After binding, washing and elution, analyse protein of interest by SDS-PAGE analysis.<br>

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=== Determining NaCl Elution Conditions  ===

'''Procedure '''<br>1. A 0.35-ml portion of 5% (w/v) PEI solution is added per 10 ml of extract as above, mixed, divided into 2-ml portions, centrifuged, and the supernatant discarded. 

2. The identical pellets are resuspended for 10 min in 2 ml of TGED buffer (50 m M Tris-HCl, pH 7.9, 5% glycerol, 0.1 m M EDTA , 0.1 mM dithiothreitol) containing various concentrations of NaCl from 0 to I M and centrifuged. 

3. Supernatants are again assayed for protein and enzyme. 

When this step is optimized, the target protein should be release from pellet into supernatant.

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=== 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]]

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