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SRF (Phospho-Ser99) Colorimetric Cell-Based ELISA Kit (CBCAB00225)

SKU:
CBCAB00225
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€960

Description

system_update_altDatasheetMSDS

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

The SRF Phospho-Ser99 Colorimetric Cell-Based ELISA Kit is specifically designed for the quantitative detection of phosphorylated Ser99 levels in cell lysates. This kit utilizes a colorimetric assay method, allowing for easy and efficient measurement of SRF phosphorylation levels in a variety of cell types.Phosphorylation of Ser99 on SRF (Serum Response Factor) is a key event in the regulation of gene expression and cell proliferation. Dysregulation of SRF signaling has been implicated in various diseases, including cancer, heart disease, and neurological disorders.

The SRF Phospho-Ser99 Colorimetric Cell-Based ELISA Kit provides researchers with a reliable tool for studying the role of SRF phosphorylation in these diseases and exploring potential therapeutic interventions.With its high sensitivity and specificity, this ELISA kit offers accurate and consistent results, making it an essential tool for researchers interested in the molecular mechanisms of cell signaling and gene expression. Order your kit today and uncover the secrets of SRF phosphorylation in cellular processes and disease pathways.

Product Name:SRF (Phospho-Ser99) Colorimetric Cell-Based ELISA
Product Code:CBCAB00225
ELISA Type:Cell-Based
Target:SRF (Phospho-Ser99)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The SRF (Phospho-Ser99) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect SRF protein 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.

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 antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. 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 absorbance values.
2. Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.
Database Information:Gene ID: 6722, UniProt ID: P11831, OMIM: 600589, Unigene: Hs.520140
Gene Symbol:SRF
Sub Type:Phospho
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
ComponentQuantity
96-Well Cell Culture Clear-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 Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 seals

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

  • Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
  • Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
  • 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)
  • Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

StepProcedure
1.Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. 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 five minutes each time with gentle shaking on the orbital shaker. 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 5 minutes each time.
9.Add 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 5 minutes each time.
11.Add 50 µL of 1x primary antibodies Anti-SRF (Phospho-Ser99) Antibody, Anti-SRF Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover 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 5 minutes each time.
13.Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)

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