Reactions
between Herceptin
Fab mutants (6 μM) bearing1 or 2 at
LC-Tyr92, LC-Thr93, or HC-Gly103 and 750 nM ErbB2 ECD were carried
out in 20 μL DPBS (adjusted to pH 8.8 with bicine) at 37 °C
for 48 h. Reactions between 1 μM Herceptin Fab-VSF92 and 4 μM
ErbB2 ECD were carried out in 20 μL DPBS, pH 7.4, at 37 °C
for 2 h, unless otherwise specified. Prior to SDS-PAGE, reactions
were quenched by boiling at 90 °C in SDS-PAGE loading buffer
supplemented with β-mercaptoethanol for 5 min. Gel band density
was determined using ImageJ software (http://rsbweb.nih.gov/ij/ ), and reaction completion was calculated as the intensity of the
cross-linked product relative to total ErbB2 (free plus cross-linked).
Cell-based cross-linking of Herceptin Fab mutants was carried out
in a 12-well plate at a density of 1 × 105 cells per
well in DMEM supplemented with 10% FBS at 37 °C and 5% CO2. At ∼70% confluency, 25 nM Herceptin Fab-VSF92 or
Herceptin Fab-WT was added to each well and incubated for 0.5, 1,
2, or 12 h. Cells were washed twice with cold DPBS and lysed using
250 μL of CelLytic M solution (Sigma) at 4 °C for 30 min.
The clarified supernatant was analyzed by Western blot with an antihuman
ErbB2 antibody (29D8, Cell Signaling Technologies) that targets the
intracellular domain of full length ErbB2.
between Herceptin
Fab mutants (6 μM) bearing
LC-Tyr92, LC-Thr93, or HC-Gly103 and 750 nM ErbB2 ECD were carried
out in 20 μL DPBS (adjusted to pH 8.8 with bicine) at 37 °C
for 48 h. Reactions between 1 μM Herceptin Fab-VSF92 and 4 μM
ErbB2 ECD were carried out in 20 μL DPBS, pH 7.4, at 37 °C
for 2 h, unless otherwise specified. Prior to SDS-PAGE, reactions
were quenched by boiling at 90 °C in SDS-PAGE loading buffer
supplemented with β-mercaptoethanol for 5 min. Gel band density
was determined using ImageJ software (
cross-linked product relative to total ErbB2 (free plus cross-linked).
Cell-based cross-linking of Herceptin Fab mutants was carried out
in a 12-well plate at a density of 1 × 105 cells per
well in DMEM supplemented with 10% FBS at 37 °C and 5% CO2. At ∼70% confluency, 25 nM Herceptin Fab-VSF92 or
Herceptin Fab-WT was added to each well and incubated for 0.5, 1,
2, or 12 h. Cells were washed twice with cold DPBS and lysed using
250 μL of CelLytic M solution (Sigma) at 4 °C for 30 min.
The clarified supernatant was analyzed by Western blot with an antihuman
ErbB2 antibody (29D8, Cell Signaling Technologies) that targets the
intracellular domain of full length ErbB2.