162
1. Prepare BGP-C10 (NH 2 -EAYRRFYGPV-COOH) and BGPC10dV (NH 2 -EAYRRFYGP-COOH) as antigen and nonantigen peptide, respectively.
2. Mix 25 μL per well of 140 nM protein samples in PBS and
25 μL per well of antigen peptides in PBS. Incubate for
30 min at room temperature in a black microplate. Use
approximately 1 μM protein stock and dilute it in PBS immediately before the assay.
3. Apply 50 μL per well of 2 μM fluorocillin in PBS containing
urea, TritonX-100, Tween-20 or BSA, at varying concentrations.
Use 1 mM fluorocillin stock solution in DMSO and dilute it in
PBS immediately before the assay.
4. Immediately read for their fluorescence intensity at 535 nm
with 485 nm at 30 °C for 2 h using a fluorescence plate reader
GENios Pro.
5. Fit the initial rates of reactions to the Michaelis-Menten equation as follows:
by the nonlinear least squares algorithm of Kaleidagraph 4.1
(Synergy Software, Reading, PA, USA).
Evaluate the catalytic activity of Cbody-cpBLA using a colorimetric substrate nitrocefin, as shown in Fig. 4.
1. Mix 25 μL per well of 140 nM proteins in PBS and 25 μL per
well of antigen samples in PBS. Incubate for 30 min at room
temperature in transparent microplate. Use approximately
1 μM stock protein and dilute this in PBS immediately before
the assay.
2. Apply 50 μL per well of 0–500 μM nitrocefin in PBS containing 2.4 M urea. Use 20 mM nitrocefin stock solution in DMSO
and dilute it in PBS immediately before the assay.
3. Immediately read for their absorbance at 486 nm at 30 °C for
1 h using a microplate reader SH-1000.
4. Calculate the catalytic parameters following the same procedures in Subheading 3.11.
4 Notes
1. SHuffle T7 Express lysY is an engineered E. coli B strain to
express proteins containing disulfide bonds in the cytoplasm.
Constitutively expressed disulfide bond isomerase DsbC
promotes the correction of mis-oxidized proteins into their
correct form, and also works as a chaperone that can assist in
the folding of proteins that do not require disulfide bonds.
3.12 Clampbody
Activity Assay
with Nitrocefin
Hiroto Iwai et al.
1. Prepare BGP-C10 (NH 2 -EAYRRFYGPV-COOH) and BGPC10dV (NH 2 -EAYRRFYGP-COOH) as antigen and nonantigen peptide, respectively.
2. Mix 25 μL per well of 140 nM protein samples in PBS and
25 μL per well of antigen peptides in PBS. Incubate for
30 min at room temperature in a black microplate. Use
approximately 1 μM protein stock and dilute it in PBS immediately before the assay.
3. Apply 50 μL per well of 2 μM fluorocillin in PBS containing
urea, TritonX-100, Tween-20 or BSA, at varying concentrations.
Use 1 mM fluorocillin stock solution in DMSO and dilute it in
PBS immediately before the assay.
4. Immediately read for their fluorescence intensity at 535 nm
with 485 nm at 30 °C for 2 h using a fluorescence plate reader
GENios Pro.
5. Fit the initial rates of reactions to the Michaelis-Menten equation as follows:
by the nonlinear least squares algorithm of Kaleidagraph 4.1
(Synergy Software, Reading, PA, USA).
Evaluate the catalytic activity of Cbody-cpBLA using a colorimetric substrate nitrocefin, as shown in Fig. 4.
1. Mix 25 μL per well of 140 nM proteins in PBS and 25 μL per
well of antigen samples in PBS. Incubate for 30 min at room
temperature in transparent microplate. Use approximately
1 μM stock protein and dilute this in PBS immediately before
the assay.
2. Apply 50 μL per well of 0–500 μM nitrocefin in PBS containing 2.4 M urea. Use 20 mM nitrocefin stock solution in DMSO
and dilute it in PBS immediately before the assay.
3. Immediately read for their absorbance at 486 nm at 30 °C for
1 h using a microplate reader SH-1000.
4. Calculate the catalytic parameters following the same procedures in Subheading 3.11.
4 Notes
1. SHuffle T7 Express lysY is an engineered E. coli B strain to
express proteins containing disulfide bonds in the cytoplasm.
Constitutively expressed disulfide bond isomerase DsbC
promotes the correction of mis-oxidized proteins into their
correct form, and also works as a chaperone that can assist in
the folding of proteins that do not require disulfide bonds.
3.12 Clampbody
Activity Assay
with Nitrocefin
Hiroto Iwai et al.