Guanylate Cyclase beta Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB00120
€499

Description

Guanylate Cyclase beta Cell-Based ELISA Kit

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

How does our Guanylate Cyclase beta Colorimetric Cell-Based ELISA Kit work?

Qualitative determination of Guanylate Cyclase beta concentration is achieved by an indirect ELISA format. In essence, Guanylate Cyclase beta is captured by Guanylate Cyclase beta -specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° 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.

Guanylate Cyclase beta Colorimetric Cell-Based ELISA Kit - Information

Product Name:Guanylate Cyclase beta Colorimetric Cell-Based ELISA Kit
Product Code:/SKUCBCAB00120
Description:The Guanylate Cyclase beta Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Guanylate Cyclase beta protein expression profile in cells. The kit can be used for measuring the relative amounts of Guanylate Cyclase beta in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on Guanylate Cyclase beta.
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse, Rat
Assay Type:Cell-Based
Database Links:Gene ID: 2983, UniProt ID: Q02153, OMIM #: 139397, Unigene #: Hs.77890
Format:96-Well Microplate
NCBI Gene Symbol:GUCY1B3
Sub Type:None
Target Name:Guanylate Cyclase beta

Kit Principle

Figure: Schematic representation of ELISA Genie Cell-Based Colorimetric ELISA principle

Kit components

Quantity

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 ul
100x Anti-Target Antibody60 ul
Anti-GAPDH Antibody60 ul
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

Additional equipment and materials required

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

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

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-Guanylate Cyclase beta 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.)

Guanylate Cyclase beta - Protein Information

UniProt Protein Function:GUCY1B3: Belongs to the adenylyl cyclase class-4/guanylyl cyclase family. Heterodimer of an alpha and a beta chain. 2 isoforms of the human protein are produced by alternative splicing
UniProt Protein Details:

Protein type:Receptor, misc.; Guanylyl cyclase; Lyase; Nucleotide Metabolism - purine; EC 4.6.1.2

Chromosomal Location of Human Ortholog: 4q31.3-q33

Cellular Component: guanylate cyclase complex, soluble; intracellular membrane-bound organelle; cytoplasm

Molecular Function:guanylate cyclase activity; GTP binding; protein heterodimerization activity; metal ion binding; heme binding; Hsp90 protein binding; receptor activity

Biological Process: cGMP biosynthetic process; blood circulation; blood coagulation; nitric oxide mediated signal transduction

NCBI Summary:This gene encodes the beta subunit of the soluble guanylate cyclase (sGC), which catalyzes the conversion of GTP (guanosine triphosphate) to cGMP (cyclic guanosine monophosphate). The encoded protein contains an HNOX domain, which serves as a receptor for ligands such as nitric oxide, oxygen and nitrovasodilator drugs. Alternative splicing results in multiple transcript variants. [provided by RefSeq, May 2014]
UniProt Code:Q02153
NCBI GenInfo Identifier:399328
NCBI Gene ID:2983
NCBI Accession:Q02153.1
UniProt Secondary Accession:Q02153,Q86WY5, B7Z426,
UniProt Related Accession:Q02153
Molecular Weight:619
NCBI Full Name:Guanylate cyclase soluble subunit beta-1
NCBI Synonym Full Names:guanylate cyclase 1, soluble, beta 3
NCBI Official Symbol:GUCY1B3  
NCBI Official Synonym Symbols:GUCB3; GC-SB3; GUC1B3; GUCSB3; GUCY1B1; GC-S-beta-1  
NCBI Protein Information:guanylate cyclase soluble subunit beta-1; GCS-beta-1; GCS-beta-3; soluble guanylate cyclase small subunit; guanylate cyclase soluble subunit beta-3
UniProt Protein Name:Guanylate cyclase soluble subunit beta-1
UniProt Synonym Protein Names:Guanylate cyclase soluble subunit beta-3; GCS-beta-3; Soluble guanylate cyclase small subunit
Protein Family:Guanylate cyclase soluble
UniProt Gene Name:GUCY1B3  
UniProt Entry Name:GCYB1_HUMAN
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