Editing 293EF Protein Production (section)

== Protein Purification<br>  ==

Components of the Expi293 Expression Medium can strip Ni-NTA resins, interfering with the ability of the affinity resin to bind polyhistidine-tagged<br>proteins. Therefore His-Assist Nickel Affinity Purification Aid (Cat. no. A14644) should be added to medium if Ni column is used for protein purification.<br> 

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Use the appropriate amount of resin for the expected yield of protein. Affinity resins have various binding capacities; Protein A Agarose from Invitrogen<br>(Cat. no. 15918-014) has a binding capacity of &gt;18 mg of human IgG per mL of gel.<br><br> 

1. Prepare resin for binding: Prepare a column containing the appropriate amount of Protein A resin to bind the amount of recombinant protein<br> produced in Expi293F culture. Pass at least 5 column volumes of 1X PBS over the column to remove the preservative solution.<br> 

<br>2. Prepare IgG-containing culture medium sample: Centrifuge the cells out of the transfected culture (10 minutes at 2000–5000 × g). Repeat if necessary.<br> Filter the culture medium through a 0.2-μm filter (optional). Supernatant (i.e., medium) can be stored at 4°C for several days or –20°C longer term.<br> 

<br>3. Bind IgG to Protein A resin: Pass the desired volume of clarified media over the prepared Protein A column.<br> 

<br>4. Wash resin: Pass at least 10 resin volumes of 1X PBS over the column. Collect the unbound protein fraction and save at 4°C or –20°C for future analysis.<br><br> 

5. Elute and recover IgG: Elute by adding at least 5 resin volumes of elution buffer and collect the fractions. Neutralize the fractions by adding 1/10th<br> volume of neutralization buffer.<br> 

<br>6. Analyze: Analyze IgG levels in the eluted samples by ELISA or SDS-PAGE. 

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'''Vector for Protein Production:'''<br>

1395 pCMV5-hIgG-Lc-TagGFP-Fc(3c)-WPRE-OriP<br>

'''Enhancer Plasmid:''' <br>1348 pLVX-EF1-VP16-EBNA1c (EF1 promoter): for stable/longterm expression<br>or 1392 pAAV-VP16-EBNA1c (for transient expression).<br>

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[[Image:293EF 4.jpg]] 

[[Image:293EF 3.jpg]] 

[[Image:293EF 2.jpg]]<br><br>[[Image:293EF 1.jpg]]<br> 

[[Category:Protocols]]