Anti Myosin, phospho Ser19/Ser20

ELISA Results of Rabbit Anti Myosin pS19/pS20 Antibody tested against BSA conjugated peptide of immunizing peptide. Each well was coated in duplicate with 0.1ug of Myosin pS19/pS20 [Red Line] and Myosin S19/S20 [Green Line]. The starting dilution of antibody was 5μg/ml and the X axis represents the Log10 of a 3 fold dilution. This titration is a 4 parameter curve fit where the IC50 is defined as the titer of the antibody. Assay performed using Goat anti Rabbit IgG Antibody Peroxidase Conjugated (Min X Bv Ch Gt GP Ham Hs Hu Ms Rt & Sh Serum Proteins) (p/n 611 103 122) and TMB ELISA Peroxidase Substrate (p/n TMBE 1000).

Rockland's affinity purified anti-Monophosphorylated RLC Smooth and Non-Muscle Myosin pS19/20 antibody was used at 2.5 ug/ml to detect signal in a variety of tissues including multi-human, multi-brain and multi-cancer slides. This image shows strong staining of both vascular and myometrial smooth muscle cells of the uterus. Tissue was formalin-fixed and paraffin embedded. The image shows localization of the antibody as the precipitated red signal, with a hematoxylin purple nuclear counterstain. Personal Communication, Tina Roush, LifeSpanBiosciences, Seattle, WA.

Immunohistochemistry with anti-myosin pS19/pS20 antibody showing strong cytoplasmic staining of myocytes in mouse heart muscle 20x and 40x (B & C). Staining was performed on Leica Bond system using the standard protocol. Formalin fixed/paraffin embedded tissue sections were subjected to antigen retrieval and then incubated with rabbit anti-myosin pS19/pS20 antibody at 1:100 dilution for 60 minutes. Biotinylated Anti-rabbit secondary antibody was used to detect primary antibody. The reaction was developed using streptavidin-HRP conjugated compact polymer system and visualized with chromogen substrate, 3’3-diamino-benzidine substrate (DAB). The sections were then counterstained with hematoxylin to detect cell nuclei.

Affinity Purified Phospho specific antibody to Monophosphorylated Regulatory Light Chain of Smooth and Non-muscle Myosin at pS19/pS20 was used at a 1:5000 dilution to detect myosin light chain by Western blot. Either 13uL or 20 ug of a mouse cardiac myocyte lysate was loaded on a 4-20% Criterion gel for SDS-PAGE. Samples were either mock-treated or CLA-treated, as indicated. After washing, a 1:5,000 dilution of HRP conjugated Gt-a-Rabbit IgG (611-103-122) preceded color development using Amersham's substrate system. Other detection methods will yield similar results. Data courtesy of the Alliance for Cellular Signaling (http://www.signaling-gateway.org).

Affinity purified phosphospecific antibody to phosphorylated regulatory light chain of smooth and non-muscle Myosin at pS19/pS20 was used at a 1:1000 dilution to detect myosin light chain by Western blot on 3T3 cell lysates. A standard urea/glycerol gel without SDS was used to separate phospho forms of regulatory light chain according to mass to charge ratios. In Panel A on the left, reactivity of Rockland's phosphospecific antibody is shown. In Panel B on the right, reactivity of commercially available pan reactive antibody that detects both un-phosphorylated and phosphorylated forms of regulatory light chain is shown. Rockland's phosphospecific antibody detects both mono-phosphorylated (pSer20 Mono-P-RLC) and di-phosphorylated (pThr19-pSer20 Di-P-RLC) regulatory light chain. Personal communication. J. Stull. UT Southwestern Medical Center.

Western blot of Rabbit Anti-Myosin pS19/pS20 primary antibody. Lane 1: Regulatory Light Chain Non-Phospho recombinant protein. Lane 2: Regulatory Light Chain Phospho recombinant protein. Lane 3: Smooth Muscle Non-Phospho recombinant protein. Lane 4: Smooth Muscle Phospho recombinant protein. Load: 50 ng per lane. Primary antibody: Myosin pS19/pS20 primary antibody at 1:1,000 overnight at 4C. Secondary antibody: Peroxidase rabbit secondary antibody at 1:40,000 for 60 min at RT. Blocking: MB-070 for 30 min at RT. Predicted/Observed size: 20 kDa, 20 kDa for Regulatory Light Chain Phospho. Other band(s): None.