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MEF2C (Phospho-Ser396) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01682
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Cell Death
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
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Description

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MEF2C (Phospho-Ser396)Colorimetric Cell-Based ELISA Kit

The MEF2C Phospho-Ser396 Colorimetric Cell-Based ELISA Kit is a powerful tool for researchers looking to accurately detect and quantify levels of MEF2C phosphorylated at serine 396 in cell lysates and tissue homogenates. This kit offers high sensitivity and specificity, providing reliable and reproducible results for a variety of research applications.MEF2C is a transcription factor that plays a critical role in various cellular processes, including differentiation, growth, and survival. Phosphorylation of MEF2C at serine 396 has been implicated in regulating its activity and function, making it a key target for studying signaling pathways and understanding disease mechanisms.

By using the MEF2C Phospho-Ser396 Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the role of MEF2C phosphorylation in cellular signaling pathways, disease progression, and potential therapeutic interventions. This kit is an essential tool for advancing research in fields such as cancer, cardiovascular diseases, and neurological disorders.

Product Name:MEF2C (Phospho-Ser396) Colorimetric Cell-Based ELISA
Product Code:CBCAB01682
ELISA Type:Cell-Based
Target:MEF2C (Phospho-Ser396)
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The MEF2C (Phospho-Ser396) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MEF2C protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated MEF2C in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on MEF2C phosphorylation.

Qualitative determination of MEF2C (Phospho-Ser396) concentration is achieved by an indirect ELISA format. In essence, MEF2C (Phospho-Ser396) is captured by MEF2C (Phospho-Ser396)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 4208, UniProt ID: Q06413, OMIM: 600662, Unigene: Hs.653394
Gene Symbol:MEF2C
Sub Type:Phospho
UniProt Protein Function:MEF2C: transcription factor of the MADS family which binds specifically to the MEF2 element present in the regulatory regions of many muscle-specific genes. May be involved in myogenesis, neurogenesis and in the development of cortical architecture. Three splice-variant isoforms have been described.
UniProt Protein Details:

Protein type:DNA-binding; Transcription factor

Chromosomal Location of Human Ortholog: 5q14.3

Cellular Component: cytoplasm; intracellular membrane-bound organelle; nuclear speck; nucleoplasm; nucleus; protein complex

Molecular Function:AT DNA binding; miRNA binding; protein binding; protein heterodimerization activity; transcription activator binding; transcription factor activity

Biological Process: B cell homeostasis; B cell proliferation; B cell receptor signaling pathway; blood vessel development; blood vessel remodeling; chondrocyte differentiation; endochondral ossification; germinal center formation; heart development; heart looping; humoral immune response; learning and/or memory; MAPKKK cascade; melanocyte differentiation; muscle cell fate determination; muscle development; myotube differentiation; negative regulation of neuron apoptosis; negative regulation of ossification; negative regulation of transcription from RNA polymerase II promoter; nervous system development; neural crest cell differentiation; neuron development; neuron differentiation; neuron migration; neuron morphogenesis during differentiation; osteoblast differentiation; platelet formation; positive regulation of B cell proliferation; positive regulation of bone mineralization; positive regulation of cardiac muscle cell proliferation; positive regulation of muscle cell differentiation; positive regulation of myoblast differentiation; positive regulation of neuron differentiation; positive regulation of osteoblast differentiation; positive regulation of skeletal muscle development; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; regulation of excitatory postsynaptic membrane potential; regulation of germinal center formation; regulation of megakaryocyte differentiation; regulation of neuron apoptosis; regulation of neurotransmitter secretion; regulation of synaptic activity; regulation of synaptic plasticity; regulation of synaptic transmission, glutamatergic; regulation of synaptogenesis; regulation of transcription, DNA-dependent; response to virus; skeletal muscle development; smooth muscle cell differentiation; ventricular cardiac muscle cell differentiation

Disease: Mental Retardation, Autosomal Dominant 20

NCBI Summary:This locus encodes a member of the MADS box transcription enhancer factor 2 (MEF2) family of proteins, which play a role in myogenesis. The encoded protein, MEF2 polypeptide C, has both trans-activating and DNA binding activities. This protein may play a role in maintaining the differentiated state of muscle cells. Mutations and deletions at this locus have been associated with severe mental retardation, stereotypic movements, epilepsy, and cerebral malformation. Alternatively spliced transcript variants have been described. [provided by RefSeq, Jul 2010]
UniProt Code:Q06413
NCBI GenInfo Identifier:2500875
NCBI Gene ID:4208
NCBI Accession:Q06413.1
UniProt Secondary Accession:Q06413,C9JMZ0, D7F7N5, F8W7V7,
UniProt Related Accession:Q06413
Molecular Weight:50,242 Da
NCBI Full Name:Myocyte-specific enhancer factor 2C
NCBI Synonym Full Names:myocyte enhancer factor 2C
NCBI Official Symbol:MEF2C  
NCBI Official Synonym Symbols:DEL5q14.3; C5DELq14.3  
NCBI Protein Information:myocyte-specific enhancer factor 2C
UniProt Protein Name:Myocyte-specific enhancer factor 2C
Protein Family:Myocyte-specific enhancer factor
UniProt Gene Name:MEF2C  
UniProt Entry Name:MEF2C_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-MEF2C (Phospho-Ser396) Antibody, Anti-MEF2C 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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