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MITF (Phospho-Ser180/73) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00424
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Epigenetics and Nuclear Signaling
Reactivity:
Human
Mouse
Detection Method:
Colorimetric
€599
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Description

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MITF (Phospho-Ser180/73)Colorimetric Cell-Based ELISA Kit

The MITF (Microphthalmia-associated transcription factor) Phospho-Ser180/73 Colorimetric Cell-Based ELISA Kit is specifically designed for the accurate detection of phosphorylated MITF levels in cell lysates. This kit offers high sensitivity and specificity, providing reliable and reproducible results for various research applications.MITF is a transcription factor that plays a critical role in melanocyte development, differentiation, and survival. Phosphorylation of MITF at serine 180 and 73 regulates its activity and function, making it an important target for studying melanoma and other pigmentation-related disorders.

By using the MITF Phospho-Ser180/73 Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the phosphorylation status of MITF and its downstream signaling pathways. This information can help further our understanding of melanoma progression and inform the development of potential targeted therapies for this deadly disease.

Product Name:MITF (Phospho-Ser180/73) Colorimetric Cell-Based ELISA
Product Code:CBCAB00424
ELISA Type:Cell-Based
Target:MITF (Phospho-Ser180/73)
Reactivity:Human, Mouse
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The MITF (Phospho-Ser180/73) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MITF protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated MITF in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on MITF phosphorylation.

Qualitative determination of MITF (Phospho-Ser180/73) concentration is achieved by an indirect ELISA format. In essence, MITF (Phospho-Ser180/73) is captured by MITF (Phospho-Ser180/73)-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: 4286, UniProt ID: O75030, OMIM: 103470/103500/156845/193510, Unigene: Hs.166017/Hs.618266
Gene Symbol:MITF
Sub Type:Phospho
UniProt Protein Function:MITF: a transcription factor that contains both basic helix-loop-helix and leucine zipper structural features. Plays a critical role in the differentiation of various cell types including neural crest- derived melanocytes, mast cells, osteoclasts and optic cup-derived retinal pigment epithelium. Two isoforms are known: the M-isoform is expressed exclusively in melanocytes, while the A-isoform has a much broader range of expression. Mutations in MITF can lead to Waardenburg syndrome. Ten alternatively spliced isoforms have been described.
UniProt Protein Details:

Protein type:Transcription factor; Oncoprotein; DNA-binding

Chromosomal Location of Human Ortholog: 3p14.2-p14.1

Cellular Component: protein complex; nucleus

Molecular Function:RNA polymerase II transcription factor activity, enhancer binding; protein dimerization activity; protein binding; chromatin binding

Biological Process: transcription from RNA polymerase II promoter; regulation of osteoclast differentiation; camera-type eye development; cell fate commitment; regulation of transcription, DNA-dependent; positive regulation of transcription, DNA-dependent; melanocyte differentiation; protein complex assembly; positive regulation of transcription from RNA polymerase II promoter; negative regulation of transcription from RNA polymerase II promoter; osteoclast differentiation; bone remodeling; regulation of cell proliferation

Disease: Waardenburg Syndrome, Type 2a; Albinism, Ocular, With Sensorineural Deafness; Melanoma, Cutaneous Malignant, Susceptibility To, 8; Tietz Syndrome

NCBI Summary:This gene encodes a transcription factor that contains both basic helix-loop-helix and leucine zipper structural features. It regulates the differentiation and development of melanocytes retinal pigment epithelium and is also responsible for pigment cell-specific transcription of the melanogenesis enzyme genes. Heterozygous mutations in the this gene cause auditory-pigmentary syndromes, such as Waardenburg syndrome type 2 and Tietz syndrome. Alternatively spliced transcript variants encoding different isoforms have been identified. [provided by RefSeq, Jul 2008]
UniProt Code:O75030
NCBI GenInfo Identifier:13124344
NCBI Gene ID:4286
NCBI Accession:O75030.2
UniProt Secondary Accession:O75030,Q14841, Q9P2V0, Q9P2V1, Q9P2V2, Q9P2Y8, B4DJL2 D3K197, E9PFN0,
UniProt Related Accession:O75030
Molecular Weight:526
NCBI Full Name:Microphthalmia-associated transcription factor
NCBI Synonym Full Names:microphthalmia-associated transcription factor
NCBI Official Symbol:MITF  
NCBI Official Synonym Symbols:MI; WS2; CMM8; WS2A; bHLHe32  
NCBI Protein Information:microphthalmia-associated transcription factor; class E basic helix-loop-helix protein 32
UniProt Protein Name:Microphthalmia-associated transcription factor
UniProt Synonym Protein Names:Class E basic helix-loop-helix protein 32; bHLHe32
Protein Family:Microphthalmia-associated transcription factor
UniProt Gene Name:MITF  
UniProt Entry Name:MITF_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-MITF (Phospho-Ser180/73) Antibody, Anti-MITF 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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