702368

antibody from Invitrogen Antibodies

Targeting: MYL9 LC20, MLC2, MRLC1, MYRL2

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

702368

Provider

Invitrogen Antibodies

Product name

Phospho-MYL9 (Thr18, Ser19) Recombinant Rabbit Monoclonal Antibody (18H3L4)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Reactivity

Human, Mouse

Host

Rabbit

Isotype

IgG

Antibody clone number

18H3L4

Vial size

100 µg

Concentration

0.5 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extract (30 µg lysate) of C2C12 (Lane 1), C2C12 treated with CalyculinA (50 nM for 20 min) (Lane 2), U87MG (Lane 3), U87MG treated with CalyculinA (50 ng for 20 min) (Lane 4). The blots were probed with Anti-Myosin regulatory light polypeptide 9 (pT18/pS19) Recombinant Rabbit Monoclonal Antibody (Product # 702368, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 20 kDa band corresponding to Myosin regulatory light polypeptide 9 (pT18/pS19) was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, C2C12 cells were fixed and permeabilized for detection of endogenous myosin regulatory light polypeptide 9 pT (18)pS (19) using Anti-myosin regulatory light polypeptide 9 pT (18)pS (19) Recombinant Rabbit Monoclonal Antibody (Product # 702368, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of myosin regulatory light polypeptide 9 pT (18)pS (19) protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of myosin regulatory light polypeptide 9 pT (18)pS (19). Panel e) shows loss of signal by competition with the myosin regulatory light polypeptide 9 pT (18)pS (19) peptide, demonstrating antibody specificity, and panel f) demonstrates no competition with the non-phospho peptide. Panel g) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.