- Dialyze the ligation mix on a 0.025μm filter paper against
sterile water for 30 min and transform into electrocompetent
XL1-Blue cells. - Cells recovered from transformation are plated on LB agar
plates containing 100μg/mL ampicillin. Pick single colonies
from transformation plates and plasmid DNA prepared by
routine technique and digest with NcoI–NotI enzymes to
check the presence of correct sized insert (~750 bp) by gel
electrophoresis. Check the insert by sequencing using primers
AH18REV and HuCκFOR. - Small scale prokaryotic expression of scAb fragments. Single
colonies of positive clones selected instep 17are inoculated
into 5 mL of LB medium containing 1% (wt/vol) glucose,
50 μg/mL ampicillin, and 12.5μg/mL tetracycline, and incu-
bated overnight at 37C with shaking. - The overnight cultures from step 18 are centrifuged at
2000 gfor 10 min and the cell pellets resuspended in 5 mL
of fresh LB containing 100μg/mL ampicillin and 12.5μg/mL
tetracycline, and grown at 25C with shaking for 1 h. - Protein expression is induced by adding IPTG to a final con-
centration of 1 mM. After IPTG addition, cells are grown for
further for 4 h and further processed as described instep 25. - Medium scale prokaryotic expression of scAb fragments. Inoc-
ulate 0.5 mL of overnight cultures of single positive clones
selected instep 17in 50 mL of TB medium containing potas-
sium phosphate buffer, 100μg/mL ampicillin, 12.5μg/mL
tetracycline, and 1% (wt/vol) glucose in 250 mL conical flasks.
Grow the cultures for 7–8 h at 37C with shaking. - Centrifuge the cultures fromstep 21at 2000gfor 15 min.
Resuspend the cell pellets in fresh TB medium as above and
incubate overnight at 30C with shaking. - The following day, collect the pellets by centrifuging at
2000 g for 20 min and resuspend in fresh TB medium
containing potassium phosphate buffer and 100μg/mL ampi-
cillin by vortexing. - Recover cells for 1 h at 25C with shaking and then induce
with IPTG as described instep 20. - Periplasmic release of expressed scAbs. After IPTG induction,
cultures fromsteps 20and 24 are centrifuged at 4000gfor
20 min, and the cell pellets resuspended in 1/10 of the original
culture volume of Fractionation buffer. Incubate on ice for
15 min with gentle shaking. To this suspension, add 1/10 of
the original culture volume of ice cold 5 mM MgSO 4 and
incubate 15 min on ice. Centrifuge at 4000gfor 20 min,
Monoclonal Antibodies Inactivating AHL Signal Molecules 349