Merlin Colorimetric Cell-Based ELISA Kit (CBCAB00063)

Product Name:	Merlin Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00063
ELISA Type:	Cell-Based
Target:	Merlin
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of Merlin concentration is achieved by an indirect ELISA format. In essence, Merlin is captured by Merlin-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: 4771, UniProt ID: P35240, OMIM: 101000/162091/607379, Unigene: Hs.187898
Gene Symbol:	NF2
Sub Type:	None

UniProt Protein Function:	Merlin: a moesin-ezrin-radizin-like protein. Interacts with cell-surface proteins, proteins involved in cytoskeletal dynamics and proteins involved in regulating ion transport. Expressed at high levels during embryonic development; in adults, significant expression is found in Schwann cells, meningeal cells, lens and nerve. Mutations are associated with neurofibromatosis type II. Two predominant isoforms and eight minor isoforms are produced by alternatively spliced transcripts.
UniProt Protein Details:	Protein type:Tumor suppressor; Motility/polarity/chemotaxis; Cytoskeletal Chromosomal Location of Human Ortholog: 22q12.2 Cellular Component: adherens junction; apical part of cell; cleavage furrow; cortical actin cytoskeleton; cytoplasm; cytoskeleton; early endosome; extrinsic to membrane; filopodium membrane; lamellipodium; lipid raft; membrane; neuron projection; nucleolus; nucleus; perinuclear region of cytoplasm; plasma membrane; protein complex; synapse Molecular Function:actin binding; beta-catenin binding; integrin binding; protein binding; protein domain specific binding Biological Process: actin cytoskeleton organization and biogenesis; ectoderm development; hippocampus development; intercellular junction assembly and maintenance; mesoderm formation; negative regulation of cell migration; negative regulation of cell proliferation; negative regulation of cell-cell adhesion; negative regulation of cell-matrix adhesion; negative regulation of DNA replication; negative regulation of JAK-STAT cascade; negative regulation of MAPKKK cascade; negative regulation of protein kinase activity; negative regulation of tyrosine phosphorylation of Stat3 protein; negative regulation of tyrosine phosphorylation of Stat5 protein; odontogenesis of dentine-containing teeth; positive regulation of cell differentiation; positive regulation of stress fiber formation; regulation of gliogenesis; regulation of protein stability; Schwann cell proliferation; small GTPase mediated signal transduction Disease: Meningioma, Familial, Susceptibility To; Neurofibromatosis, Type Ii; Schwannomatosis 1
NCBI Summary:	This gene encodes a protein that is similar to some members of the ERM (ezrin, radixin, moesin) family of proteins that are thought to link cytoskeletal components with proteins in the cell membrane. This gene product has been shown to interact with cell-surface proteins, proteins involved in cytoskeletal dynamics and proteins involved in regulating ion transport. This gene is expressed at high levels during embryonic development; in adults, significant expression is found in Schwann cells, meningeal cells, lens and nerve. Mutations in this gene are associated with neurofibromatosis type II which is characterized by nervous system and skin tumors and ocular abnormalities. Two predominant isoforms and a number of minor isoforms are produced by alternatively spliced transcripts. [provided by RefSeq, Jul 2008]
UniProt Code:	P35240
NCBI GenInfo Identifier:	462594
NCBI Gene ID:	4771
NCBI Accession:	P35240.1
UniProt Secondary Accession:	P35240,O95683, Q8WUJ2, Q969N0, Q969Q3, Q96T30, Q96T31 Q96T32, Q96T33, Q9BTW3, Q9UNG9, Q9UNH3,
UniProt Related Accession:	P35240
Molecular Weight:	24,515 Da
NCBI Full Name:	Merlin
NCBI Synonym Full Names:	neurofibromin 2 (merlin)
NCBI Official Symbol:	NF2
NCBI Official Synonym Symbols:	ACN; SCH; BANF
NCBI Protein Information:	merlin
UniProt Protein Name:	Merlin
UniProt Synonym Protein Names:	Moesin-ezrin-radixin-like protein; Neurofibromin-2; Schwannomerlin; Schwannomin
Protein Family:	Merlin
UniProt Gene Name:	NF2
UniProt Entry Name:	MERL_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-Merlin 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.)