Opioid Receptor-delta (Phospho-Ser363) Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB01266
Product Type:
ELISA Kit
ELISA Type:
Cell Based Phospho Specific
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
Frequently bought together:

Description

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Opioid Receptor-delta (Phospho-Ser363)Colorimetric Cell-Based ELISA Kit

The Opioid Receptor Delta Phospho-Ser363 Colorimetric Cell-Based ELISA Kit from Assaygenie is a cutting-edge tool designed for the accurate measurement of phospho-ser363 levels in opioid receptor delta in cell cultures. This kit offers high sensitivity and specificity, ensuring precise and reproducible results, making it an essential tool for researchers studying opioid receptor delta signaling pathways and potential therapeutic interventions.Opioid receptor delta is a key player in pain modulation and addiction processes, making it a valuable target for drug development and understanding the mechanisms of opioid action.

By detecting phospho-ser363 modification, researchers can gain insights into the regulatory mechanisms of opioid receptor delta and its potential implications in various physiological and pathological conditions.Overall, the Opioid Receptor Delta Phospho-Ser363 Colorimetric Cell-Based ELISA Kit provides researchers with a reliable platform to investigate the role of opioid receptor delta in health and disease, opening up new possibilities for drug discovery and therapeutic interventions in the field of neuroscience and pain management.

Product Name:Opioid Receptor-delta (Phospho-Ser363) Colorimetric Cell-Based ELISA
Product Code:CBCAB01266
ELISA Type:Cell-Based
Target:Opioid Receptor-delta (Phospho-Ser363)
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Format:2 x 96-Well Microplates

The Opioid Receptor-delta (Phospho-Ser363) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Opioid Receptor-delta protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Opioid Receptor-delta in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Opioid Receptor-delta phosphorylation.

Qualitative determination of Opioid Receptor-delta (Phospho-Ser363) concentration is achieved by an indirect ELISA format. In essence, Opioid Receptor-delta (Phospho-Ser363) is captured by Opioid Receptor-delta (Phospho-Ser363)-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: 4985, UniProt ID: P41143, OMIM: 165195, Unigene: Hs.372
Gene Symbol:OPRD1
Sub Type:Phospho
UniProt Protein Function:DOR-1: delta opioid receptor, a G-protein coupled receptor. Inhibits neurotransmitter release by reducing calcium ion currents and increasing potassium ion conductance. Highly stereoselective. Receptor for enkephalins.
UniProt Protein Details:

Protein type:GPCR, family 1; Membrane protein, multi-pass; Receptor, GPCR; Membrane protein, integral

Chromosomal Location of Human Ortholog: 1p36.1-p34.3

Cellular Component: cytoplasm; integral to plasma membrane; intrinsic to plasma membrane; lipid raft; nerve terminal; neuron projection; plasma membrane; postsynaptic membrane; vesicle

Molecular Function:neuropeptide binding; opioid receptor activity; protein binding

Biological Process: activation of CREB transcription factor; adult locomotory behavior; eating behavior; G-protein coupled receptor protein signaling pathway; G-protein signaling, adenylate cyclase inhibiting pathway; G-protein signaling, coupled to cyclic nucleotide second messenger; G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); immune response; negative regulation of protein oligomerization; neuropeptide signaling pathway; positive regulation of peptidyl-serine phosphorylation; protein import into nucleus, translocation; regulation of calcium ion transport; regulation of mitochondrial membrane potential; regulation of sensory perception of pain; synaptic transmission

UniProt Code:P41143
NCBI GenInfo Identifier:311033488
NCBI Gene ID:4985
NCBI Accession:P41143.4
UniProt Secondary Accession:P41143,B5B0B8,
UniProt Related Accession:P41143
Molecular Weight:40,369 Da
NCBI Full Name:Delta-type opioid receptor
NCBI Synonym Full Names:opioid receptor delta 1
NCBI Official Symbol:OPRD1  
NCBI Official Synonym Symbols:OPRD  
NCBI Protein Information:delta-type opioid receptor
UniProt Protein Name:Delta-type opioid receptor
Protein Family:Delta-type opioid receptor
UniProt Gene Name:OPRD1  
UniProt Entry Name:OPRD_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-Opioid Receptor-delta (Phospho-Ser363) Antibody, Anti-Opioid Receptor-delta 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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