Horseradish peroxidase conjugated anti mouse igg

To assess CaM-dependent and independent enzymatic activity of smMLCK, the construct used in AFM experiments was incubated with human RLC (NP_291024.1) in the presence and absence of Ca²⁺/CaM and the phosphorylation of RLC serine 19 was probed.
10 nM smMLCK was mixed with 10 µM RLC, 500 µM ATP in 20 mM HEPES (pH 7.5), 50 mM NaCl, 10 mM MgCl ${}_2$ , 1 mM DTT, 500 µM ATP in the presence of either 40 nM CaM/1 mM CaCl ${}_2$ or 1 mM EGTA and incubated at 20°C. Samples were taken at various intervals with the reaction quenched by addition of SDS-PAGE loading buffer.
Samples containing 300 ng RLC were run on SDS-PAGE alongside BioRad Precision Plus protein marker, transferred onto nitrocellulose membrane, incubated in 5% milk at room temperature for 45 min and probed with Cell Signaling Technology antibody 3671 against phospo-myosin light chain 2 (Ser19) in 5% milk for 2 hr at room temperature. The membrane was washed with low-salt buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 0.1% Tween-20) three times and then incubated with horseradish peroxidase conjugated anti-mouse IgG (Dako P0260) in 5% milk for 45 min. After washing three times in low-salt buffer the membrane was stained using the enhanced chemiluminescence method.

ELISAs were performed between indirectly coated HLA I/peptide complexes and phage clones or Fab antibodies [32 (link)]. Plate-bound streptavidin (5 µg/ml) was incubated with biotinylated HLA I/peptide complexes at 4 µg/ml. To confirm correct folding of HLA I/peptide complexes, we used the conformation-specific monoclonal antibody Tü155 (kindly provided by A. Ziegler, Berlin, Germany). Fab antibodies were incubated with indirectly coated HLA I/peptide complexes at a concentration of 10 µg/ml for 1 h at room temperature. Fab binding was confirmed using the murine anti-myc antibody 9E10 (Roche, Mannheim, Germany) and a horseradish peroxidase-conjugated anti-mouse IgG (Dako, Glostrup, Denmark). Bound phages were detected using the murine IgG antibody M13 (Amersham Pharmacia Biotech, Sweden).

Anti-IRE1α was prepared as follows. cDNA encoding the cytosolic domain (1513–3054) corresponding to amino acids 465–977 of mouse IRE1α (GenBank Accession No. NM023913) was amplified using pcDNA3.1 (+) mIRE1α 33 (link) as a template and 5′-ACATGCATGCACTTACCCCCTGAGCGTG-3′ and 5′-ACGCGTCGACTCAGAGGGCATATGGAATCACT-3′ as primers. The amplified DNA was digested with SphI and SalI and cloned into a pQE80L vector (Qiagen, Hilden, Germany) to produce the His-tagged protein in Escherichia coli. Antigens were purified as previously described34 (link). For immunization, a rabbit was injected with an emulsion containing 0.2 mg antigen and Sigma Adjuvant System (Sigma-Aldrich, St. Louis, MO), for 4 times with 3-week intervals between injections; antiserum was collected at 10 days after the final injection. Anti-C/EBP homologous protein (CHOP), anti-eukaryotic initiation factor 2α (eIF2α), anti-phospho-eIF2α, and anti-β-actin were purchased from Affinity BioReagents/Thermo Fisher Scientific (Waltham, MA), Cell Signaling Technology (Danvers, MA), Biosource (Camarillo, CA), and Novus Biologicals (Littleton, CO), respectively. Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from DAKO (Glostrup, Denmark) and GE Healthcare (Little Chalfont, UK), respectively.