SRF (Phospho-Ser99) Fluorometric Cell-Based ELISA Kit

SKU:

FBCAB00015

Product Type:

ELISA Kit

ELISA Type:

Cell Based Phospho Specific

Reactivity:

Human

Mouse

Rat

Detection Method:

Fluorometric

Description

Description

SRF (Phospho-Ser99) Cell-Based ELISA Kit

The SRF (Phospho-Ser99) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor SRF phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated SRF in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on SRF phosphorylation.

How does our SRF (Phospho-Ser99) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of SRF (Phospho-Ser99) concentration is achieved by an indirect ELISA format. In essence, SRF (Phospho-Ser99) is captured by SRF (Phospho-Ser99)-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 SRF (Phospho-Ser99) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated SRF can be used to normalize the RFU value for phosphorylated SRF.

SRF (Phospho-Ser99) Fluorometric Cell-Based ELISA Kit -Information

Product Name:	SRF (Phospho-Ser99) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:	FBCAB00015
Description:	The SRF (Phospho-Ser99) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor SRF (Phospho-Ser99) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated SRF (Phospho-Ser99) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on SRF 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: 6722, UniProt ID: P11831, OMIM #: 600589, Unigene #: Hs.520140
Format:	Two 96-Well Plates
NCBI Gene Symbol:	SRF
Sub Type:	Phospho
Target Name:	Phospho-SRF (Ser99)

Kit Principle

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

Kit components	Quantity
96-Well Black Cell CultureClear-Bottom Microplate	2 plates
10X TBS	24 ml
Quenching Buffer	24 ml
Blocking Buffer	50 ml
15X Wash Buffer	50 ml
Primary Antibody Diluent	12 ml
100x Anti-Phospho Target Antibody	60 µl
100x Anti-Target Antibody	60 µl
Anti-GAPDH Antibody	110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody	6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody	6 ml
Adhesive Plate Seals	2 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 SRF (Phospho-Ser99) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total SRF 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.

SRF (Phospho-Ser99) - Protein Information

UniProt Protein Function:	SRF: a transcription factor of the MADS domain family that binds to the serum response element (SRE). Regulates the transcription of immediate early genes including c-fos. Binds DNA as a multimer, probably a dimer.
UniProt Protein Details:	Protein type:Transcription factor; Motility/polarity/chemotaxis Chromosomal Location of Human Ortholog: 6p21.1 Cellular Component: cytoplasm; nuclear chromatin; nucleoplasm; nucleus Molecular Function:chromatin DNA binding; histone deacetylase binding; protein binding; protein homodimerization activity; RNA polymerase II transcription factor activity, enhancer binding; transcription factor activity; transcription factor binding Biological Process: angiogenesis involved in wound healing; associative learning; cardiac myofibril assembly; cell migration during sprouting angiogenesis; cell-matrix adhesion; developmental growth; erythrocyte development; heart development; heart looping; hippocampus development; long-term memory; mesoderm formation; morphogenesis of an epithelial sheet; mRNA transcription from RNA polymerase II promoter; muscle maintenance; negative regulation of cell migration; negative regulation of cell proliferation; neurite development; neuron development; neuron migration; patterning of blood vessels; platelet activation; platelet formation; positive regulation of axon extension; positive regulation of cell differentiation; positive regulation of filopodium formation; positive regulation of smooth muscle contraction; positive regulation of transcription by glucose; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; positive thymic T cell selection; regulation of cell adhesion; regulation of smooth muscle cell differentiation; regulation of water loss via skin; response to cytokine stimulus; response to hormone stimulus; response to hypoxia; response to toxin; sarcomere organization; skin morphogenesis; small GTPase mediated signal transduction; stress fiber formation; tangential migration from the subventricular zone to the olfactory bulb; thymus development; thyroid gland development; transcription from RNA polymerase II promoter; trophectodermal cell differentiation
NCBI Summary:	This gene encodes a ubiquitous nuclear protein that stimulates both cell proliferation and differentiation. It is a member of the MADS (MCM1, Agamous, Deficiens, and SRF) box superfamily of transcription factors. This protein binds to the serum response element (SRE) in the promoter region of target genes. This protein regulates the activity of many immediate-early genes, for example c-fos, and thereby participates in cell cycle regulation, apoptosis, cell growth, and cell differentiation. This gene is the downstream target of many pathways; for example, the mitogen-activated protein kinase pathway (MAPK) that acts through the ternary complex factors (TCFs). Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, May 2014]
UniProt Code:	P11831
NCBI GenInfo Identifier:	134876
NCBI Gene ID:	6722
NCBI Accession:	P11831.1
UniProt Secondary Accession:	P11831,Q5T648,
UniProt Related Accession:	P11831
Molecular Weight:	51,593 Da
NCBI Full Name:	Serum response factor
NCBI Synonym Full Names:	serum response factor
NCBI Official Symbol:	SRF
NCBI Official Synonym Symbols:	MCM1
NCBI Protein Information:	serum response factor
UniProt Protein Name:	Serum response factor
Protein Family:	Serum response factor
UniProt Gene Name:	SRF
UniProt Entry Name:	SRF_HUMAN