Goat anti mouse igg horseradish peroxidase conjugate

For immunoblot analysis pneumococcal cell lysates 5x10 8 bacteria were loaded per lane. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane by semidry blotting (Bio-Rad Laboratories, Munich, Germany). The membrane was blocked with 5% skim milk (Roth) in Tris buffered saline (TBS).
Detection of PnrA was conducted with specific mouse anti-PnrA antibodies and goat anti-mouse IgG horseradish peroxidase conjugate (Dianova, Hamburg, Germany; 1:5000) as described [21] . The loading control protein enolase was detected with rabbit anti-enolase antibodies and a goat anti-rabbit IgG horseradish peroxidase conjugate (1:5000). Enhanced chemiluminescence (luminol and p-coumaric acid, Roth) was used for signal detection.

Bacterial subfractionated extracts, as well as the positive control recombinant SPINK9-v1, were loaded onto a 12% SDS-tricine polyacrylamide gel containing 8 M urea. Proteins were transferred to a Protran-nitrocellulose membrane (Schleicher & Schuell BioScience, Dassel, Germany), blocked for 1 hour in blocking buffer (5% (w/v) nonfat powdered milk in phosphate buffered saline þ 0.05% Tween), then incubated for 18 hours at 4 C in 3% (w/v) nonfat powdered milk in phosphate buffered saline þ 0.05% Tween containing 1:1,250 affinity-purified polyclonal anti-SPINK9 antibody. The membrane was washed with phosphate buffered saline þ 0.05% Tween six times for 5 minutes each, then incubated for 1 hour in 3% (w/v) nonfat powdered milk in phosphate buffered saline þ 0.05% Tween containing 1:20,000 dilution of goat anti-mouse IgG horseradish peroxidase conjugate (Dianova, Hamburg, Germany). After six times washing steps as before the membrane was incubated for 5 minutes with chemiluminescent peroxidase substrate (Sigma, Taufkirchen, Germany) and visualized using a Diana III cooled CCDcamera imaging system (Raytest, Straubenhardt, Germany).
Densitometric quantifications were performed using AIDA evaluation software (Raytest).