Myosin regulatory light chain 2 (Phospho-Ser18) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00043
Product type:
ELISA Kit
ELISA Type:
Cell Based
€599

Description

Technical Manual

Myosin regulatory light chain 2 (Phospho-Ser18) Cell-Based ELISA Kit

The Myosin regulatory light chain 2 (Phospho-Ser18) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor Myosin regulatory light chain 2 phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Myosin regulatory light chain 2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Myosin regulatory light chain 2 phosphorylation.

How does our Myosin regulatory light chain 2 (Phospho-Ser18) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of Myosin regulatory light chain 2 (Phospho-Ser18) concentration is achieved by an indirect ELISA format. In essence, Myosin regulatory light chain 2 (Phospho-Ser18) is captured by Myosin regulatory light chain 2 (Phospho-Ser18)-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target RFU values.
2. An antibody against the nonphosphorylated counterpart of Myosin regulatory light chain 2 (Phospho-Ser18) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated Myosin regulatory light chain 2 can be used to normalize the RFU value for phosphorylated Myosin regulatory light chain 2.

Myosin regulatory light chain 2 (Phospho-Ser18) Fluorometric Cell-Based ELISA Kit -Information

Product Name:Myosin regulatory light chain 2 (Phospho-Ser18) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00043
Description:The Myosin regulatory light chain 2 (Phospho-Ser18) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor Myosin regulatory light chain 2 (Phospho-Ser18) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Myosin regulatory light chain 2 (Phospho-Ser18) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on MLRN phosphorylation.
Dynamic Range:> 5000 Cells
Detection Method:Fluorometric
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse, Rat
Assay Type:Cell-Based ELISA
Database Links:Gene ID: 3566, UniProt ID: P24844, OMIM #: 147781, Unigene #: Hs.513457
Format:Two 96-Well Plates
NCBI Gene Symbol:MLRN
Sub Type:Phospho
Target Name:Phospho-Myosin regulatory light chain 2 (Ser18)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components

Quantity

96-Well Black Cell CultureClear-Bottom Microplate2 plates
10X TBS24 ml
Quenching Buffer24 ml
Blocking Buffer50 ml
15X Wash Buffer50 ml
Primary Antibody Diluent12 ml
100x Anti-Phospho Target Antibody 60 µl
100x Anti-Target Antibody60 µl
Anti-GAPDH Antibody110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody6 ml
Adhesive Plate Seals2 seals

Additional equipment and materials required

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
  • Micropipettes capable of measuring volumes from 1 µl to 1 ml
  • Deionized or sterile water (ddH2O)
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

1.Seed 200 µl of desired cell concentration in culture medium into each well of the 96-well plates. For suspension cells and loosely attached cells, coat the plates with 100 µl of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µl of 1x TBS, twice.
5.Fix the cells by incubating with 100 µl of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µl 1x Wash Buffer for 3 minutes. The plate can be stored at 4°C for a week.
7.Add 100 µl of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 3 minutes each time.
9.Dispense 200 µl of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
11.Add 50 µl of Primary Antibody Mixture P to corresponding wells for Myosin regulatory light chain 2 (Phospho-Ser18) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total Myosin regulatory light chain 2 detection. Cover the plate with parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
13.Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

Myosin regulatory light chain 2 (Phospho-Ser18) - Protein Information

UniProt Protein Function:MRLC1: 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. Myosin is an hexamer of 2 heavy chains and 4 light chains. Smooth muscle tissues and in some, but not all, nonmuscle cells. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Contractile; Motility/polarity/chemotaxis

Chromosomal Location of Human Ortholog: 20q11.23

Cellular Component: cytosol; muscle myosin complex; stress fiber; Z disc

Molecular Function:calcium ion binding; myosin heavy chain binding; structural constituent of muscle

Biological Process: axon guidance; ephrin receptor signaling pathway; muscle contraction; regulation of muscle contraction; small GTPase mediated signal transduction

NCBI Summary:Myosin, a structural component of muscle, consists of two heavy chains and four light chains. The protein encoded by this gene is a myosin light chain that may regulate muscle contraction by modulating the ATPase activity of myosin heads. The encoded protein binds calcium and is activated by myosin light chain kinase. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:P24844
NCBI GenInfo Identifier:20141521
NCBI Gene ID:10398
NCBI Accession:P24844.4
UniProt Secondary Accession:P24844,Q9BQL9, Q9BUF9, Q9H136, E1P5T6,
UniProt Related Accession:P24844
Molecular Weight:13,866 Da
NCBI Full Name:Myosin regulatory light polypeptide 9
NCBI Synonym Full Names:myosin light chain 9
NCBI Official Symbol:MYL9  
NCBI Official Synonym Symbols:LC20; MLC2; MRLC1; MYRL2; MLC-2C  
NCBI Protein Information:myosin regulatory light polypeptide 9
UniProt Protein Name:Myosin regulatory light polypeptide 9
UniProt Synonym Protein Names:20 kDa myosin light chain; LC20; MLC-2C; Myosin RLC; Myosin regulatory light chain 2, smooth muscle isoform; Myosin regulatory light chain 9; Myosin regulatory light chain MRLC1
Protein Family:Myosin regulatory light polypeptide
UniProt Gene Name:MYL9  
UniProt Entry Name:MYL9_HUMAN