mGluR4 Colorimetric Cell-Based ELISA Kit (CBCAB00754)

Product Name:	mGluR4 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00754
ELISA Type:	Cell-Based
Target:	mGluR4
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The mGluR4 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect mGluR4 protein expression profile in cells. The kit can be used for measuring the relative amounts of mGluR4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on mGluR4.

Qualitative determination of mGluR4 concentration is achieved by an indirect ELISA format. In essence, mGluR4 is captured by mGluR4-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: 2914, UniProt ID: Q14833, OMIM: 604100, Unigene: Hs.654847
Gene Symbol:	GRM4
Sub Type:	None

UniProt Protein Function:	mGluR4: metabotropic glutamate receptor 3, a G-protein coupled receptor for glutamate. L-glutamate is the major excitatory neurotransmitter in the central nervous system and activates both ionotropic and metabotropic glutamate receptors. The activity of this receptor is mediated by a G-protein that inhibits adenylate cyclase activity.
UniProt Protein Details:	Protein type:Membrane protein, integral; Cell development/differentiation; Membrane protein, multi-pass; Receptor, GPCR; GPCR, family 3 Chromosomal Location of Human Ortholog: 6p21.3 Cellular Component: presynaptic membrane; integral to plasma membrane; plasma membrane; terminal button; presynaptic active zone membrane; intracellular; cytoplasmic vesicle; dendritic shaft Molecular Function:calmodulin binding; G-protein coupled receptor activity; group III metabotropic glutamate receptor activity; glutamate receptor activity; calcium-dependent protein binding Biological Process: synaptic transmission; regulation of synaptic transmission, glutamatergic; positive regulation of MAPKKK cascade; activation of MAPK activity; neurotransmitter secretion; regulation of neuron apoptosis; learning; metabotropic glutamate receptor, adenylate cyclase inhibiting pathway
NCBI Summary:	L-glutamate is the major excitatory neurotransmitter in the central nervous system and activates both ionotropic and metabotropic glutamate receptors. Glutamatergic neurotransmission is involved in most aspects of normal brain function and can be perturbed in many neuropathologic conditions. The metabotropic glutamate receptors are a family of G protein-coupled receptors, that have been divided into 3 groups on the basis of sequence homology, putative signal transduction mechanisms, and pharmacologic properties. Group I includes GRM1 and GRM5 and these receptors have been shown to activate phospholipase C. Group II includes GRM2 and GRM3 while Group III includes GRM4, GRM6, GRM7 and GRM8. Group II and III receptors are linked to the inhibition of the cyclic AMP cascade but differ in their agonist selectivities. Several transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Feb 2012]
UniProt Code:	Q14833
NCBI GenInfo Identifier:	2495077
NCBI Gene ID:	2914
NCBI Accession:	Q14833.1
UniProt Secondary Accession:	Q14833,Q5SZ84, Q6ZMQ2, B3KVL9, B7Z1T9, B7Z1U6, F5GXM5
UniProt Related Accession:	Q14833,AAB34891
Molecular Weight:
NCBI Full Name:	Metabotropic glutamate receptor 4
NCBI Synonym Full Names:	glutamate receptor, metabotropic 4
NCBI Official Symbol:	GRM4
NCBI Official Synonym Symbols:	mGlu4; GPRC1D; MGLUR4
NCBI Protein Information:	metabotropic glutamate receptor 4
UniProt Protein Name:	Metabotropic glutamate receptor 4
Protein Family:	Metabotropic glutamate receptor
UniProt Gene Name:	GRM4
UniProt Entry Name:	GRM4_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-mGluR4 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.)