GluR1 Colorimetric Cell-Based ELISA Kit (CBCAB00304)

Product Name:	GluR1 Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00304
ELISA Type:	Cell-Based
Target:	GluR1
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The GluR1 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GluR1 protein expression profile in cells. The kit can be used for measuring the relative amounts of GluR1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on GluR1.

Qualitative determination of GluR1 concentration is achieved by an indirect ELISA format. In essence, GluR1 is captured by GluR1-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: 2890, UniProt ID: P42261, OMIM: 138248, Unigene: Hs.519693
Gene Symbol:	GRIA1
Sub Type:	None

UniProt Protein Function:	GluR1: an integral membrane protein belonging to the glutamate-gated ion channel family. L-glutamate (Glu) acts as an excitatory neurotransmitter at many synapses in the central nervous system. Glutamate receptors are heteromeric protein complexes with multiple subunits, each possessing transmembrane regions, and all arranged to form a ligand-gated ion channel. The postsynaptic actions of Glu are mediated by a variety of receptors that are named according to their selective agonists. Each of the four GluR proteins (GRIA1-4) include flip and flop isoforms generated by alternative RNA splicing. Expressed in granule and pyramidal cells in the hippocampal formation.
UniProt Protein Details:	Protein type:Membrane protein, integral; Membrane protein, multi-pass; Channel, calcium; Channel, ligand-gated Chromosomal Location of Human Ortholog: 5q31.1 Cellular Component: cell junction; cell soma; cell surface; dendrite; dendritic spine; endoplasmic reticulum membrane; ER-Golgi intermediate compartment membrane; Golgi membrane; plasma membrane; postsynaptic density; postsynaptic membrane; recycling endosome; synaptic vesicle Molecular Function:alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate selective glutamate receptor activity; extracellular-glutamate-gated ion channel activity; glutamate receptor activity; PDZ domain binding; protein binding Biological Process: cellular protein metabolic process; COPII coating of Golgi vesicle; ER to Golgi vesicle-mediated transport; ionotropic glutamate receptor signaling pathway; long-term memory; post-translational protein modification; protein amino acid N-linked glycosylation via asparagine; receptor internalization; signal transduction; synaptic transmission
NCBI Summary:	Glutamate receptors are the predominant excitatory neurotransmitter receptors in the mammalian brain and are activated in a variety of normal neurophysiologic processes. These receptors are heteromeric protein complexes with multiple subunits, each possessing transmembrane regions, and all arranged to form a ligand-gated ion channel. The classification of glutamate receptors is based on their activation by different pharmacologic agonists. This gene belongs to a family of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors. Alternatively spliced transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P42261
NCBI GenInfo Identifier:	116242505
NCBI Gene ID:	2890
NCBI Accession:	P42261.2
UniProt Secondary Accession:	P42261,Q2NKM6, B7Z2S0, B7Z2W8, B7Z3F6, B7Z9G9, D3DQI4 E7ESV8,
UniProt Related Accession:	P42261
Molecular Weight:	102,864 Da
NCBI Full Name:	Glutamate receptor 1
NCBI Synonym Full Names:	glutamate ionotropic receptor AMPA type subunit 1
NCBI Official Symbol:	GRIA1
NCBI Official Synonym Symbols:	GLUH1; GLUR1; GLURA; GluA1; HBGR1
NCBI Protein Information:	glutamate receptor 1
UniProt Protein Name:	Glutamate receptor 1
UniProt Synonym Protein Names:	AMPA-selective glutamate receptor 1; GluR-A; GluR-K1; Glutamate receptor ionotropic, AMPA 1; GluA1
UniProt Gene Name:	GRIA1
UniProt Entry Name:	GRIA1_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-GluR1 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.)