MYF6 Colorimetric Cell-Based ELISA (CBCAB00989)

Product Name:	MYF6 Colorimetric Cell-Based ELISA
Product Code:	CBCAB00989
ELISA Type:	Cell-Based
Target:	MYF6
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The MYF6 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect MYF6 protein expression profile in cells. The kit can be used for measuring the relative amounts of MYF6 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on MYF6.

Qualitative determination of MYF6 concentration is achieved by an indirect ELISA format. In essence, MYF6 is captured by MYF6-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: 4618, UniProt ID: P23409, OMIM: 159991/160150, Unigene: Hs.35937
Gene Symbol:	MYF6
Sub Type:	None

UniProt Protein Function:	Myf-6: Involved in muscle differentiation (myogenic factor). Induces fibroblasts to differentiate into myoblasts. Probable sequence specific DNA-binding protein. Defects in MYF6 may be a cause of centronuclear myopathy type 3 (CNM3). A congenital muscle disorder characterized by progressive muscular weakness and wasting involving mainly limb girdle, trunk, and neck muscles. It may also affect distal muscles. Weakness may be present during childhood or adolescence or may not become evident until the third decade of life. Ptosis is a frequent clinical feature. The most prominent histopathologic features include high frequency of centrally located nuclei in muscle fibers not secondary to regeneration, radial arrangement of sarcoplasmic strands around the central nuclei, and predominance and hypotrophy of type 1 fibers.
UniProt Protein Details:	Protein type:DNA-binding; Transcription factor Chromosomal Location of Human Ortholog: 12q21 Cellular Component: nucleoplasm; nucleus Molecular Function:RNA polymerase II transcription factor activity, enhancer binding; protein heterodimerization activity; transcription factor activity Biological Process: regulation of transcription from RNA polymerase II promoter; transcription from RNA polymerase II promoter; muscle cell differentiation; skeletal muscle development; somitogenesis; positive regulation of skeletal muscle fiber development; muscle cell fate commitment; positive regulation of muscle cell differentiation; skeletal muscle regeneration; positive regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; positive regulation of myoblast differentiation Disease: Myopathy, Centronuclear, 3
NCBI Summary:	The protein encoded by this gene is a probable basic helix-loop-helix (bHLH) DNA binding protein involved in muscle differentiation. The encoded protein likely acts as a heterodimer with another bHLH protein. Defects in this gene are a cause of autosomal dominant centronuclear myopathy (ADCNM). [provided by RefSeq, May 2010]
UniProt Code:	P23409
NCBI GenInfo Identifier:	4505299
NCBI Gene ID:	4618
NCBI Accession:	NP_002460.1
UniProt Secondary Accession:	P23409,Q53X80, Q6FHI9, B2R898,
UniProt Related Accession:	P23409
Molecular Weight:
NCBI Full Name:	myogenic factor 6
NCBI Synonym Full Names:	myogenic factor 6 (herculin)
NCBI Official Symbol:	MYF6
NCBI Official Synonym Symbols:	CNM3; MRF4; myf-6; bHLHc4
NCBI Protein Information:	myogenic factor 6
UniProt Protein Name:	Myogenic factor 6
UniProt Synonym Protein Names:	Class C basic helix-loop-helix protein 4; bHLHc4; Muscle-specific regulatory factor 4
Protein Family:	Myogenic factor
UniProt Gene Name:	MYF6
UniProt Entry Name:	MYF6_HUMAN

Component	Quantity
96-Well Cell Culture Clear-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	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 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.

Step	Procedure
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-MYF6 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.)