Detection of in vivo phosphorylated proteins in crude extracts of S. pneumoniae strains was performed after SDS-PAGE by immunoblotting using an anti-phosphothreonine polyclonal antibody (Cell Signaling) at 1/2000 as described in [14] (link). A goat anti-rabbit secondary antibody HRP conjugate (Biorad) was used at 1/5000. Detection of StkP and GFP fusions were performed using a rabbit polyclonal antibody specific for StkP [14] (link) and rabbit anti-GFP (AMS Biotechnology).
Anti phosphothreonine polyclonal antibody
Manufactured by Cell Signaling Technology
The Anti-phosphothreonine polyclonal antibody is a laboratory reagent used for the detection and study of proteins containing phosphorylated threonine residues. This antibody is produced by immunizing animals with a synthetic phosphothreonine-containing peptide and purifying the resulting polyclonal antibodies.
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4 protocols using anti phosphothreonine polyclonal antibody
1
Detecting Phosphorylated Proteins in S. pneumoniae
2
Immunoblotting for MapZ Phosphorylation
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After protein separation by 4–12% gradient SDS-PAGE, detection of MapZ phosphorylation in crude extracts of S. pneumoniae WT and pmp23 mutant strains was performed by immunoblotting using an anti-phosphothreonine polyclonal antibody (Cell Signaling) at 1/2,000 as described previously44 (link). A goat anti-rabbit secondary antibody HRP conjugate (Biorad) was used at 1/5,000 to reveal the immunoblots.
3
Immunodetection of Phosphorylated Proteins in S. pneumoniae
4
In Vitro Phosphorylation Assay of Recombinant Proteins
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In vitro phosphorylation was carried out with 0.5 μg of purified recombinant substrate protein and 0.5 μg of purified GST-StkP in 30 μl of kinase buffer (50 mM Tris-HCl, 5 mM MgCl2, 100 μM ATP, 1mM DTT, pH 7.5). The reaction was started by the addition of ATP and stopped after 60 min of incubation at 37°C by the addition of 5x Laemmli SDS sample buffer. Samples were separated by standard Tris-glycine-SDS polyacrylamide gel electrophoresis (PAGE) gels and electroblotted onto a nitrocellulose membrane. Phosphorylated proteins were detected with an anti-phosphothreonine polyclonal antibody (1∶1,000; Cell Signaling) and a goat anti-rabbit immunoglobulin G secondary antibody conjugated to horseradish peroxidase (1∶2,500; Invitrogen). Detection was performed with an enhanced chemiluminescence substrate (SuperSignal West Pico Chemiluminescent Substrate; Pierce) and Hyperfilm CL film (GE) using exposures of between 1 and 10 min. The pRSET-divIVAspn plasmid was generously provided by Dr. Orietta Massidda (Università degli studi di Cagliari, Italy) [78 (link)].
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