IL-10Ralpha Colorimetric Cell-Based ELISA Kit

(No reviews yet) Write a Review
SKU:
CBCAB00710
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Detection Method:
Colorimetric

Description

system_update_altDatasheet
Product Name:IL-10Ralpha Colorimetric Cell-Based ELISA
Product Code:CBCAB00710
ELISA Type:Cell-Based
Target:IL-10Ralpha
Reactivity:Human
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

The IL-10Ralpha Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect IL-10Ralpha protein expression profile in cells. The kit can be used for measuring the relative amounts of IL-10Ralpha in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on IL-10Ralpha.

Qualitative determination of IL-10Ralpha concentration is achieved by an indirect ELISA format. In essence, IL-10Ralpha is captured by IL-10Ralpha-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: 3587, UniProt ID: Q13651, OMIM: 146933/613148, Unigene: Hs.504035
Gene Symbol:IL10RA
Sub Type:None
UniProt Protein Function:IL10RA: a receptor for interleukin 10 structurally related to interferon receptors. Mediates the immunosuppressive signal of interleukin 10, and thus inhibits the synthesis of proinflammatory cytokines. This receptor is reported to promote survival of progenitor myeloid cells through the insulin receptor substrate-2/PI 3-kinase/AKT pathway. Activation of this receptor leads to tyrosine phosphorylation of JAK1 and TYK2 kinases.
UniProt Protein Details:

Protein type:Membrane protein, integral; Receptor, cytokine

Chromosomal Location of Human Ortholog: 11q23

Cellular Component: integral to membrane; plasma membrane

Molecular Function:interleukin-10 binding; interleukin-10 receptor activity; protein binding; receptor activity; signal transducer activity

Biological Process: cytokine and chemokine mediated signaling pathway; response to lipopolysaccharide

Disease: Inflammatory Bowel Disease 28, Autosomal Recessive

NCBI Summary:The protein encoded by this gene is a receptor for interleukin 10. This protein is structurally related to interferon receptors. It has been shown to mediate the immunosuppressive signal of interleukin 10, and thus inhibits the synthesis of proinflammatory cytokines. This receptor is reported to promote survival of progenitor myeloid cells through the insulin receptor substrate-2/PI 3-kinase/AKT pathway. Activation of this receptor leads to tyrosine phosphorylation of JAK1 and TYK2 kinases. Two transcript variants, one protein-coding and the other not protein-coding, have been found for this gene. [provided by RefSeq, Jan 2009]
UniProt Code:Q13651
NCBI GenInfo Identifier:322510034
NCBI Gene ID:3587
NCBI Accession:Q13651.2
UniProt Secondary Accession:Q13651,A8K6I0, B0YJ27,
UniProt Related Accession:Q13651
Molecular Weight:
NCBI Full Name:Interleukin-10 receptor subunit alpha
NCBI Synonym Full Names:interleukin 10 receptor subunit alpha
NCBI Official Symbol:IL10RA  
NCBI Official Synonym Symbols:CD210; IL10R; CD210a; CDW210A; HIL-10R; IL-10R1  
NCBI Protein Information:interleukin-10 receptor subunit alpha
UniProt Protein Name:Interleukin-10 receptor subunit alpha
UniProt Synonym Protein Names:CDw210a; Interleukin-10 receptor subunit 1; IL-10R subunit 1; IL-10R1; CD_antigen: CD210
Protein Family:Interleukin-10 receptor
UniProt Gene Name:IL10RA  
UniProt Entry Name:I10R1_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-IL-10Ralpha 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.)

View AllClose

0 Reviews

View AllClose

Related Products