Rabbit polyclonal to Myosin light chain (phospho S20)
This phospho specific polyclonal antibody is specific for phosphorylated pS20 of human myosin light chain. Reactivity with non-phosphorylated human myosin light chain is less than 1% by ELISA. The selected peptide sequence used to generate the polyclonal antibody is located near the amino terminal end of the polypeptide corresponding to the smooth/non-muscle form of myosin regulatory light chain found in cardiac myocytes in addition to smooth and non-muscle cells. This sequence differs from that of the sarcomeric/cardiac form of myosin regulatory light chain that has a different sequence around the phosphorylation site. BLAST search analysis was used to determine that the smooth and non-muscle forms of myosin regulatory light chain have identical sequences. Cross reactivity is expected.
This antibody was affinity purified from monospecific antiserum by immunoaffinity purification. Antiserum was first purified against the phosphorylated form of the immunizing peptide. The resultant affinity purified antibody was then cross-adsorbed against the non-phosphorylated form of the immunizing peptide.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 2.5 µg/ml.
1/5000 - 1/20000.
1/1000 - 1/5000. Predicted molecular weight: 20 kDa. We recommend using BSA for blocking when using phospho-specific antibodies.
Use at an assay dependent concentration. PubMed: 19279134
Trichloroacetic acid was used for tissue fixation.
Use at an assay dependent concentration.
Myosin regulatory subunit that plays an important role in regulation of both smooth muscle and nonmuscle cell contractile activity via its phosphorylation. Implicated in cytokinesis, receptor capping, and cell locomotion.
Smooth muscle tissues and in some, but not all, nonmuscle cells.
Contains 3 EF-hand domains.
Phosphorylation increases the actin-activated myosin ATPase activity and thereby regulates the contractile activity. It is required to generate the driving force in the migration of the cells but not necessary for localization of myosin-2 at the leading edge.
Western blot - Anti-Myosin light chain (phospho S20) antibody (ab2480)
Rabbit polyclonal to phospho Myosin Light Chain phospho Myosin Light Chain (Ser 20) (ab2480) used at a 1/5000 dilution to detect myosin light chain by Western blot. Either 13 ug (lanes 1-3) or 20 ug (lanes 4-7) 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: Lane 1 : untreated 45 min Lane 2 : CLA 50 nm 45 min Lane 3 : CLA 100 nm 45 min Lane 4 : A21 untreated 45 min Lane 5 : A22 A23187 5 min Lane 6 : A23 A23187 15 min Lane 7 : A24 A23187 60 min After washing, a 1/5,000 dilution of anti-rabbit HRP (ab7090) was used as secondary.
Rabbit polyclonal to phospho Myosin Light Chain phospho Myosin Light Chain (Ser 20) (ab2480) used at a 1/5000 dilution to detect myosin light chain by Western blot. Either 13 ug (lanes 1-3) or 20 ug (lanes 4-7) of a mouse cardiac myocyte lysate was loaded on a 4-20
IHC-P of ab2480 at 2.5 µg/ml staining both vascular and myometrial smooth muscle cells of the uterus. The image shows localization of the antibody as the precipitated red signal, with a hematoxylin purple nuclear counterstain.
Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Myosin light chain (phospho S20) antibody (ab2480)Image from Tao Shijie et. al., Development 136, 1327-1338 (2009), Fig 7G.
ab2480 staining Myosin light chain (phospho S20) in Xenopus laevis embryos by immunohistochemistry (PFA-perfusion fixed frozen tissue sections). The tissue sections were fixed in 2% TCA for 30 minutes and washed with PBST (PBS+0.3% TritonX 100) for 30 minutes. Samples were then blocked in 10% normal goat serum (NGS) for 1 hour at room temperature and incubated with the primary antibody. A Cy5 labelled Goat polyclonal to rabbit IgG was used as secondary.
The arrows shows that depleting E-cadherin does not have any effect on the expression of apical pMLC.
Immunocytochemistry/ Immunofluorescence - Anti-Myosin light chain (phospho S20) antibody (ab2480)Image from Barnhart EL et al., PLoS Biol. 2011 May;9(5):e1001059. doi: 10.1371/journal.pbio.1001059. Epub 2011 May 3. Fig 6.; May 3, 2011 PLoS Biology 9(5): e1001059.
Immunofluroescence analysis of Nicaragua cichlid (Hypsophrys nicaraguensis) keratocytes, staining Myosin light chain (phospho S20) (green) with ab2480.
Cells were cultured on intermediate adhesion strength surfaces and extracted with 4% PEG and 1% Triton X-100 in cytoskeleton stabilizing buffer. Cells were blocked with PBS-BT for 5 minutes, and then incubated with primary antibody diluted in PBS-BT. Cells were then fixed with 4% formaldehyde in PBS for 10 minutes.
Cervero P et al. Lymphocyte-specific protein 1 regulates mechanosensory oscillation of podosomes and actin isoform-based actomyosin symmetry breaking. Nat Commun9:515 (2018).
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Shinoda T et al. Elasticity-based boosting of neuroepithelial nucleokinesis via indirect energy transfer from mother to daughter. PLoS Biol16:e2004426 (2018).
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