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GRK1 (Phospho-Ser21) Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB01427
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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GRK1 (Phospho-Ser21)Colorimetric Cell-Based ELISA Kit

The GRK1 Phospho-Ser21 Colorimetric Cell-Based ELISA Kit is specifically designed for the accurate detection of phosphorylated GRK1 (G protein-coupled receptor kinase 1) levels in cell lysates. This innovative kit offers high sensitivity and specificity, ensuring precise and reproducible results for researchers in various fields.GRK1 is a critical enzyme involved in the regulation of G protein-coupled receptor signaling, playing a key role in cellular communication and signal transduction. Phosphorylation of GRK1 at Ser21 is especially important for its activity and function.

Dysregulation of GRK1 phosphorylation has been linked to various diseases, including cardiovascular disorders, neurological conditions, and cancer.By using the GRK1 Phospho-Ser21 Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the role of phosphorylated GRK1 in disease development and progression. This kit is a valuable tool for investigating signaling pathways, drug development, and identifying potential therapeutic targets related to GRK1 phosphorylation.

Product Name:GRK1 (Phospho-Ser21) Colorimetric Cell-Based ELISA
Product Code:CBCAB01427
ELISA Type:Cell-Based
Target:GRK1 (Phospho-Ser21)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The GRK1 (Phospho-Ser21) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GRK1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated GRK1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on GRK1 phosphorylation.

Qualitative determination of GRK1 (Phospho-Ser21) concentration is achieved by an indirect ELISA format. In essence, GRK1 (Phospho-Ser21) is captured by GRK1 (Phospho-Ser21)-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: 6011, UniProt ID: Q15835, OMIM: 180381/258100, Unigene: Hs.103501
Gene Symbol:GRK1
Sub Type:Phospho
UniProt Protein Function:GRK1: a Ser/Thr of the GRK family. Phosphorylates and inactivates photon-activated rhodopsin. LOF mutations lead to type 2 Oguchi disease, a form of night blindness. A link between RHOK and retinitis pigmentosa is now thought to be unlikely
UniProt Protein Details:

Protein type:Protein kinase, Ser/Thr (non-receptor); Protein kinase, AGC; EC 2.7.11.14; Kinase, protein; AGC group; GRK family; GRK subfamily

Chromosomal Location of Human Ortholog: 13q34

Molecular Function:G-protein coupled receptor kinase activity; rhodopsin kinase activity; ATP binding; protein kinase activity

Biological Process: rhodopsin mediated signaling; post-embryonic retina morphogenesis in camera-type eye; phototransduction, visible light; regulation of rhodopsin mediated signaling; visual perception; protein amino acid autophosphorylation; photoreceptor cell morphogenesis; regulation of G-protein coupled receptor protein signaling pathway; positive regulation of phosphorylation; negative regulation of apoptosis

Disease: Oguchi Disease 2

NCBI Summary:This gene encodes a member of the guanine nucleotide-binding protein (G protein)-coupled receptor kinase subfamily of the Ser/Thr protein kinase family. The protein phosphorylates rhodopsin and initiates its deactivation. Defects in GRK1 are known to cause Oguchi disease 2 (also known as stationary night blindness Oguchi type-2). [provided by RefSeq, Jul 2008]
UniProt Code:Q15835
NCBI GenInfo Identifier:2833269
NCBI Gene ID:6011
NCBI Accession:Q15835.1
UniProt Secondary Accession:Q15835,Q53X14,
UniProt Related Accession:Q15835
Molecular Weight:563
NCBI Full Name:Rhodopsin kinase
NCBI Synonym Full Names:G protein-coupled receptor kinase 1
NCBI Official Symbol:GRK1  
NCBI Official Synonym Symbols:RK; RHOK; GPRK1  
NCBI Protein Information:rhodopsin kinase
UniProt Protein Name:Rhodopsin kinase
UniProt Synonym Protein Names:G protein-coupled receptor kinase 1
UniProt Gene Name:GRK1  
UniProt Entry Name:RK_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-GRK1 (Phospho-Ser21) Antibody, Anti-GRK1 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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