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2016-06-13T15:24:32Z

Created page with "This protocol is not working 9. Taq DNA polymerase can be recovered from this clarified lysat..."

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9. Taq DNA polymerase can be recovered from this clarified lysate by

*Precipitation with polyethyleneimine (PEI), Neutralized 10% PEI (Miles) at a final concentration of 0.05 to 0.8%, usually 0.15% or 0.6% (should be titrated using 200 ul aliquots). Precipitate can be resuspended in 200 ul '''Buffer C''' (20 mM HEPES, pH 7.9, 0.1 mM EDTA, 0.5% Tween 20,  0.25 M KCl to elute the DNA polymerase. After spining, the supernatant can be either mixed with solid ammonium sulfate to 50% saturation to precipitate Taq DNA polymerase,  or directly used for next step purification.

'''Preparing Neutralized 5% (w/v) PEI Stock Solution''': One hundred milliliters 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.

*Precipitation with solid ammonium sulfate to 50% saturation to precipitate Taq DNA polymerase. The pellet can be dissolved in Ni washing buffer.

<s>10. While mixing using stirrer bar,  slowly add solid dibasic potassium phosphate powder to the clarified lysate to a final concentration of 10%.</s>

<s>11. Add equal volume of ethanol to the 10% salt solution and mixed vigorously for several seconds, spin for 5 min at 6000 rpm.</s>

<s>12. Carefully remove the bottom aqueous phase containing nucleic acid. Collect and upper orgainc phase containing Taq DNA polymerase. Do not collect the interface containing denatured protein.</s>

<s>13. Load the orgainc phase to a Ni column. Wash column with Ni washing buffer. Elute Taq DNA polymerase using Buffer B containing 150 mM Imidazole.</s>
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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.

'''To further purify PEI-precipitated Taq Polymerase using BioRex 70:'''

1. Dilute the PEI eluate to 50 mM KCl or NaCl with '''Buffer D '''(20 mM HEPES, pH 7.9,  0.5% Tween 20)

2. Apply to a 150-ml column of BioRex 70 ion exchanger (Bio-Rad) that had been preequilibrated in '''Buffer E''' (20 mM HEPES, pH 7.9, 0.5% Tween 20, '''50''' mM KCl or NaCl).

3. Wash with 6 column volumes of '''Buffer E'''

4. Elute the enzyme with '''Buffer F''' (20 mM HEPES, pH 7.9,  0.1% Tween 20, '''200''' mM KCl).

5. Pool the fractions containing the peak of protein

6. Dialyzed for 12 h against two changes of 1 liter each '''Storage Buffer''' (20 mM HEPES, pH 7.9, 100 mM KCl, 0.1 mM EDTA, 0.5 mM PMSF, 1 mM dithiothreitol, 50% glycerol).

[[Category:Protocols]]

PEI Precipitation of Taq - Revision history

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