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GluR4 (Phospho-Ser862) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01426
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Research Area:
Signal Transduction
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

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GluR4 (Phospho-Ser862)Colorimetric Cell-Based ELISA Kit

The Human GluR4 Phospho-Ser862 Colorimetric Cell-based ELISA Kit is a powerful tool for measuring the levels of phosphorylated GluR4 at Ser862 in cell lysates. This kit offers high sensitivity and specificity, providing accurate and reproducible results for research applications.GluR4, a subunit of the AMPA-type glutamate receptor, plays a crucial role in synaptic plasticity and neuronal excitability. Phosphorylation at Ser862 has been linked to receptor trafficking, synaptic plasticity, and neuroprotective effects, making it a key target for studying neurological disorders and potential therapeutic interventions.

With this ELISA kit, researchers can gain valuable insights into the mechanisms underlying neuronal function and dysfunction, paving the way for new discoveries in neuroscience and drug development.

Product Name:GluR4 (Phospho-Ser862) Colorimetric Cell-Based ELISA
Product Code:CBCAB01426
ELISA Type:Cell-Based
Target:GluR4 (Phospho-Ser862)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The GluR4 (Phospho-Ser862) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GluR4 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated GluR4 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on GluR4 phosphorylation.

Qualitative determination of GluR4 (Phospho-Ser862) concentration is achieved by an indirect ELISA format. In essence, GluR4 (Phospho-Ser862) is captured by GluR4 (Phospho-Ser862)-specific primary (1ø) antibodies while the HRP-conjugated secondary (2ø) antibodies bind the Fc region of the 1ø antibody. Through this binding, the HRP enzyme conjugated to the 2ø 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: 2893, UniProt ID: P48058, OMIM: 138246, Unigene: Hs.503743
Gene Symbol:GRIA4
Sub Type:Phospho
UniProt Protein Function:GluR4: 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. This receptor binds AMPA(quisqualate) > glutamate > kainate. Each of the four GluR proteins (GRIA1-4) include flip and flop isoforms generated by alternative RNA splicing.
UniProt Protein Details:

Protein type:Membrane protein, integral; Channel, ligand-gated; Membrane protein, multi-pass

Chromosomal Location of Human Ortholog: 11q22

Cellular Component: cell junction; cell soma; dendrite; plasma membrane; postsynaptic membrane; terminal button

Molecular Function:alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate selective glutamate receptor activity; extracellular-glutamate-gated ion channel activity; ionotropic glutamate receptor activity

Biological Process: glutamate signaling pathway; ionotropic glutamate receptor signaling pathway; synaptic transmission; transport

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 composed of multiple subunits, arranged to form ligand-gated ion channels. The classification of glutamate receptors is based on their activation by different pharmacologic agonists. The subunit encoded by this gene belongs to a family of AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate)-sensitive glutamate receptors, and is subject to RNA editing (AGA->GGA; R->G). Alternative splicing of this gene results in transcript variants encoding different isoforms, which may vary in their signal transduction properties. Some haplotypes of this gene show a positive association with schizophrenia. [provided by RefSeq, Jul 2008]
UniProt Code:P48058
NCBI GenInfo Identifier:218512059
NCBI Gene ID:2893
NCBI Accession:P48058.2
UniProt Secondary Accession:P48058,Q86XE8,
UniProt Related Accession:P48058
Molecular Weight:49,146 Da
NCBI Full Name:Glutamate receptor 4
NCBI Synonym Full Names:glutamate ionotropic receptor AMPA type subunit 4
NCBI Official Symbol:GRIA4  
NCBI Official Synonym Symbols:GLUR4; GLURD; GluA4; GLUR4C  
NCBI Protein Information:glutamate receptor 4
UniProt Protein Name:Glutamate receptor 4
UniProt Synonym Protein Names:AMPA-selective glutamate receptor 4; GluR-D; Glutamate receptor ionotropic, AMPA 4; GluA4
Protein Family:Glutamate receptor
UniProt Gene Name:GRIA4  
UniProt Entry Name:GRIA4_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-GluR4 (Phospho-Ser862) Antibody, Anti-GluR4 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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