Adrenergic Receptor alpha-2C Colorimetric Cell-Based ELISA

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SKU:
CBCAB00983
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
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Description

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Adrenergic Receptor alpha-2C Colorimetric Cell-Based ELISA

The Adrenergic Receptor Alpha 2C Colorimetric Cell-Based ELISA Kit is a cutting-edge tool designed for the accurate measurement of alpha 2C adrenergic receptor levels in cell lysates. This innovative kit offers high sensitivity and specificity, ensuring precise and consistent results for a variety of research applications.The alpha 2C adrenergic receptor is a key player in regulating various physiological processes, including neurotransmission and vascular tone. Dysregulation of this receptor has been linked to conditions such as hypertension, anxiety disorders, and ADHD, making it a valuable target for drug development and therapeutic interventions.

With the Adrenergic Receptor Alpha 2C Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the role of this receptor in health and disease, paving the way for new treatment strategies and clinical advancements in the field of pharmacology and neuroscience.

Product Name:Adrenergic Receptor alpha-2C Colorimetric Cell-Based ELISA
Product Code:CBCAB00983
ELISA Type:Cell-Based
Target:Adrenergic Receptor alpha-2C
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The Adrenergic Receptor alpha-2C Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Adrenergic Receptor alpha-2C protein expression profile in cells. The kit can be used for measuring the relative amounts of Adrenergic Receptor alpha-2C in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Adrenergic Receptor alpha-2C.

Qualitative determination of Adrenergic Receptor alpha-2C concentration is achieved by an indirect ELISA format. In essence, Adrenergic Receptor alpha-2C is captured by Adrenergic Receptor alpha-2C-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: 152, UniProt ID: P18825, OMIM: 104250, Unigene: Hs.123022
Gene Symbol:ADRA2C
Sub Type:None
UniProt Protein Function:ADRA2C: Alpha-2 adrenergic receptors mediate the catecholamine- induced inhibition of adenylate cyclase through the action of G proteins. Belongs to the G-protein coupled receptor 1 family. Adrenergic receptor subfamily. ADRA2C sub-subfamily.
UniProt Protein Details:

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

Chromosomal Location of Human Ortholog: 4p16

Cellular Component: cytoplasm; endosome; integral to plasma membrane; plasma membrane

Molecular Function:alpha-2A adrenergic receptor binding; alpha2-adrenergic receptor activity; epinephrine binding; protein binding; protein heterodimerization activity; protein homodimerization activity

Biological Process: activation of protein kinase B; cell-cell signaling; G-protein coupled receptor protein signaling pathway; positive regulation of neuron differentiation; regulation of insulin secretion

NCBI Summary:Alpha-2-adrenergic receptors are members of the G protein-coupled receptor superfamily. They include 3 highly homologous subtypes: alpha2A, alpha2B, and alpha2C. These receptors have a critical role in regulating neurotransmitter release from sympathetic nerves and from adrenergic neurons in the central nervous system. The mouse studies revealed that both the alpha2A and alpha2C subtypes were required for normal presynaptic control of transmitter release from sympathetic nerves in the heart and from central noradrenergic neurons. The alpha2A subtype inhibited transmitter release at high stimulation frequencies, whereas the alpha2C subtype modulated neurotransmission at lower levels of nerve activity. This gene encodes the alpha2C subtype, which contains no introns in either its coding or untranslated sequences. [provided by RefSeq, Jul 2008]
UniProt Code:P18825
NCBI GenInfo Identifier:20141211
NCBI Gene ID:152
NCBI Accession:P18825.2
UniProt Secondary Accession:P18825,P35369, Q9HB49,
UniProt Related Accession:P18825
Molecular Weight:49,522 Da
NCBI Full Name:Alpha-2C adrenergic receptor
NCBI Synonym Full Names:adrenoceptor alpha 2C
NCBI Official Symbol:ADRA2C  
NCBI Official Synonym Symbols:ADRA2L2; ADRARL2; ADRA2RL2; ALPHA2CAR  
NCBI Protein Information:alpha-2C adrenergic receptor
UniProt Protein Name:Alpha-2C adrenergic receptor
UniProt Synonym Protein Names:Alpha-2 adrenergic receptor subtype C4; Alpha-2C adrenoreceptor; Alpha-2C adrenoceptor; Alpha-2CAR
Protein Family:Alpha-2C adrenergic receptor
UniProt Gene Name:ADRA2C  
UniProt Entry Name:ADA2C_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-Adrenergic Receptor alpha-2C 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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