Whole-cell protein extracts were prepared in urea-SDS sample buffer
as described previously.13 (link) SDS-polyacrylamide
gel electrophoresis (PAGE) and immunoblotting conditions to polyvinylidene
difluoride membrane (PVDF, Immobilon-P; Millipore) have been reported.13 (link),15 (link) For immunodetection, the PVDF membranes were incubated with anti-myc
mAb clone 9B11 (1:2000; Cell Signaling Technology) to detect myc-tagged
SAs and anti-mouse IgG-peroxidase (POD) conjugate (1:5000; Sigma)
as secondary antibody. GroEL was detected with anti-GroEL mAb-POD
conjugate (1:5000; Sigma). Membranes were developed by chemiluminescence
using a mixture in 100 mM Tris-HCl (pH 8.0) containing 1.25 mM luminol
(Sigma), 0.22 mM cumaric acid (Sigma), and 0.0075% (v/v) H2O2 (Sigma) and exposed to an X-ray film (Curix, Agfa).
as described previously.13 (link) SDS-polyacrylamide
gel electrophoresis (PAGE) and immunoblotting conditions to polyvinylidene
difluoride membrane (PVDF, Immobilon-P; Millipore) have been reported.13 (link),15 (link) For immunodetection, the PVDF membranes were incubated with anti-myc
mAb clone 9B11 (1:2000; Cell Signaling Technology) to detect myc-tagged
SAs and anti-mouse IgG-peroxidase (POD) conjugate (1:5000; Sigma)
as secondary antibody. GroEL was detected with anti-GroEL mAb-POD
conjugate (1:5000; Sigma). Membranes were developed by chemiluminescence
using a mixture in 100 mM Tris-HCl (pH 8.0) containing 1.25 mM luminol
(Sigma), 0.22 mM cumaric acid (Sigma), and 0.0075% (v/v) H2O2 (Sigma) and exposed to an X-ray film (Curix, Agfa).
