Editing 293F Protein Production (section)

=== Materials<br><br>  ===

==== Cell Culture<br>  ====

1. HEK-293 cells adapted to cultivation in serum-free suspension<br>2. Cylindrical and square-shaped glass bottles with nominal volumes of 100 mL to 5 L<br>3. Ex-cell 293 medium without L-glutamine and phenol red (Sigma-Aldrich).<br>4. 50× L-glutamine and phenol red solution. A stock solution with 200 mM glutamine and 250 g/mL phenol red is made by dissolving 29.23 g/L glutamine&nbsp; and 250 mg phenol red (Sigma-Aldrich) in 800 mL water. After the compounds are dissolved, the volume is adjusted to 1 L by further addition of water. The solution is sterilized by filtration through a 0.2- um Steritop (bottle-top) filter unit&nbsp; with a neck size of 45 mm (Millipore). The solution is transferred into sterile 50-mL centrifuge tubes and kept frozen at −20°C. For each liter of Ex-cell® 293 medium, 20 mL of the stock solution is added.<br>5. Trypan blue solution (0.4%) (Sigma-Aldrich).<br><br> 

==== <br>Transfection<br><br>  ====

1. RPMI 1640 medium containing 25 mM HEPES and 4 mM glutamine (Lonza).<br>2. Pluronic® F-68 dissolved in water at a concentration of 2%. The solution is filter sterilized and stored at room temperature.<br>3. VPA (2-propyl-pentanoic acid, sodium salt) (Sigma-Aldrich) is prepared as a 500-mM solution in water, filter sterilized, aliquoted into sterile 50-mL tubes, and stored at −20°C. The working solution is maintained at 4°C. VPA is a known teratogen.<br> 

4. Linear 25 kDa polyethyleneimine (PEI) (Polysciences) is dissolved in water at 1 mg/mL at a pH of 7.0. When dissolving, lower the pH with 1 N HCl. When the PEI is in solution, the pH is increased to 7.0 with&nbsp; 1 N NaOH. The solution is filter sterilized, aliquoted into sterile 50-mL tubes, and stored at −20°C. It can be stored frozen for years as long as repeated freeze–thaw cycles are avoided.<br>5. Ex-cell® 293 medium without L-glutamine and phenol red <br>6. 50× L-glutamine–phenol red solution.<br><br> 

==== IgG Purification<br><br>  ====

1. Streamline-rProtein A beads (GE Healthcare).<br>2. Econo-Pac chromatography column (Bio-Rad).<br>3. Equilibration and washing buffer: 20 mM sodium phosphate (pH 7.5).<br>4. Elution and regeneration buffer: 100 mM sodium citrate (pH 3).<br>5. Neutralizing buffer: 1 M Tris–HCl (pH 8.0).<br>6. Storage buffer: 20% ethanol in 20 mM sodium phosphate (pH 7.5).<br>7. Centricon Plus-70 (Millipore).<br>8. Phosphate buffered saline (PBS) at pH 7.1.<br><br> 

==== ELISA<br>  ====

<br>1. 96-well ELISA microtiter plates with flat bottom.<br>2. Blocking buffer: 0.5% casein hydrolysate (Applichem) and 0.05% Tween 20 (Sigma-Aldrich) in PBS (pH 7.1).<br>3. Capture antibody: Goat anti-human kappa light chain (AbD Serotec).<br>4. Coating solution: For each 96-well plate, 11 L of capture antibody is mixed with 11 mL of PBS (pH 8.0).<br>5. Washing buffer: PBS (pH 8.0) with 2% Tween 20.<br>6. Detection antibody: Alkaline phosphatase-conjugated goat anti-human gamma heavy chain (Biosource).<br>7. Standard: Human IgG, whole molecule (ChromPure, Jackson ImmunoResearch). The standard is diluted in blocking buffer to 40 ng/mL and then serially<br>diluted 1:2 with blocking buffer.<br>8. Substrate buffer: Add 97 mL diethanolamine (Sigma-Aldrich) to 700 mL H2O and adjust pH to 9.8 with 2 M HCl. Add 0.5 mL 1 M MgCl2 and 2 g NaN3. Adjust volume to 1 L.<br>9. Substrate: 4-nitrophenyl phosphate disodium salt (NPP) (Applichem) is dissolved in substrate buffer to 1.5 mg/mL.<br>10. Stop solution: 3 M NaOH.<br> 

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