4. Streptavidin (SA) Dip and Read Biosensors (Pall, Fortebio).
5. 96-well flat bottom polypropylene microplate, black.
6. PBS (1Â): 138 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 ,
2mM KH 2 PO 4 , pH7.4).
7. Kinetics buffer (1Â PBS, pH 7.4) optionally with 0.1% BSA
and 0.2% Tween 20.
8. 100 mM 10Â glycine buffer pH 1.5.
3 Methods
Carry out all procedures at room temperature unless otherwise
specified. Use sterile equipment, flasks, and centrifuge tubes
throughout the experiment.
3.1 Transient Protein
Expression
in HEK293-6E Cells
The protocol is described in Karste et al. [1]. The employed vectors
contain the OriP to ensure optimal long-term expression levels.
Here the expression is described for 500 mL.
1. [Day 3, e.g., Friday] Prepare a 400 mL culture containing 0.3
 10
6 HEK293-6E c/mL. Incubate the culture for 72 h at
37
C, 110 rpm, and 5% CO 2 .
2. [Day 0, e.g., Monday] Count the cells of the preparatory culture
and prepare a 250 mL culture containing 1.8 Â 10
6 HEK2936E c/mL by centrifuging the required volume of the cell
suspension at 180 Â g for 4 min. Discard the supernatant
carefully and resolve the cell pellet in 250 mL fresh F17. Place
the cells back into the incubator (37
C, 90 rpm, and 5% CO 2 )
until use (see Note 1).
3. Prepare the DNA solution in 12.5 mL F17 in a 50-mL corning
flask (see Note 2). Hereto, mix 237 μg of the target plasmid
(s) containing your gene(s) of interest with 13 μg control
plasmid pTTo-GFPq coding, e.g., for eGFP. Mix by agitating
the tube briefly.
4. Prepare the PEI_25 solution in 12.5 mL F17 in a 50-mL
Corning flask (see Note 2). Hereto, pipette 625 μL PEI of
the 1 mg/mL PEI_25 stock solution into the. Mix by vortexing briefly.
5. Mix the PEI_25 solution with the DNA solution directly (see
Note 3).
6. Incubate the PEI_25–DNA solution for 15 min at RT to
preform PEI–DNA complexes.
7. Pipette the PEI–DNA complexes (25 mL) to the cells and mix
gently. Incubate the cells until harvest at 37
C, 90 rpm, and 5%
CO 2 .
Expression of Recombinant Antibodies in Different Formats
65
5. 96-well flat bottom polypropylene microplate, black.
6. PBS (1Â): 138 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 ,
2mM KH 2 PO 4 , pH7.4).
7. Kinetics buffer (1Â PBS, pH 7.4) optionally with 0.1% BSA
and 0.2% Tween 20.
8. 100 mM 10Â glycine buffer pH 1.5.
3 Methods
Carry out all procedures at room temperature unless otherwise
specified. Use sterile equipment, flasks, and centrifuge tubes
throughout the experiment.
3.1 Transient Protein
Expression
in HEK293-6E Cells
The protocol is described in Karste et al. [1]. The employed vectors
contain the OriP to ensure optimal long-term expression levels.
Here the expression is described for 500 mL.
1. [Day 3, e.g., Friday] Prepare a 400 mL culture containing 0.3
 10
6 HEK293-6E c/mL. Incubate the culture for 72 h at
37
C, 110 rpm, and 5% CO 2 .
2. [Day 0, e.g., Monday] Count the cells of the preparatory culture
and prepare a 250 mL culture containing 1.8 Â 10
6 HEK2936E c/mL by centrifuging the required volume of the cell
suspension at 180 Â g for 4 min. Discard the supernatant
carefully and resolve the cell pellet in 250 mL fresh F17. Place
the cells back into the incubator (37
C, 90 rpm, and 5% CO 2 )
until use (see Note 1).
3. Prepare the DNA solution in 12.5 mL F17 in a 50-mL corning
flask (see Note 2). Hereto, mix 237 μg of the target plasmid
(s) containing your gene(s) of interest with 13 μg control
plasmid pTTo-GFPq coding, e.g., for eGFP. Mix by agitating
the tube briefly.
4. Prepare the PEI_25 solution in 12.5 mL F17 in a 50-mL
Corning flask (see Note 2). Hereto, pipette 625 μL PEI of
the 1 mg/mL PEI_25 stock solution into the. Mix by vortexing briefly.
5. Mix the PEI_25 solution with the DNA solution directly (see
Note 3).
6. Incubate the PEI_25–DNA solution for 15 min at RT to
preform PEI–DNA complexes.
7. Pipette the PEI–DNA complexes (25 mL) to the cells and mix
gently. Incubate the cells until harvest at 37
C, 90 rpm, and 5%
CO 2 .
Expression of Recombinant Antibodies in Different Formats
65