Glucagon Colorimetric Cell-Based ELISA Kit (CBCAB00675)

Product Name:	Glucagon Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00675
ELISA Type:	Cell-Based
Target:	Glucagon
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of Glucagon concentration is achieved by an indirect ELISA format. In essence, Glucagon is captured by Glucagon-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: 2641, UniProt ID: P01275, OMIM: 138030, Unigene: Hs.516494
Gene Symbol:	GCG
Sub Type:	None

UniProt Protein Function:	GCG: Glucagon plays a key role in glucose metabolism and homeostasis. Regulates blood glucose by increasing gluconeogenesis and decreasing glycolysis. A counterregulatory hormone of insulin, raises plasma glucose levels in response to insulin-induced hypoglycemia. Plays an important role in initiating and maintaining hyperglycemic conditions in diabetes. Glucagon release is stimulated by hypoglycemia and inhibited by hyperglycemia, insulin, and somatostatin. GLP-1 and GLP-2 are induced in response to nutrient ingestion. Glucagon is secreted in the A cells of the islets of Langerhans. GLP-1, GLP-2, oxyntomodulin and glicentin are secreted from enteroendocrine cells throughout the gastrointestinal tract. GLP1 and GLP2 are also secreted in selected neurons in the brain. Belongs to the glucagon family.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Secreted; Hormone Chromosomal Location of Human Ortholog: 2q36-q37 Cellular Component: extracellular space; endoplasmic reticulum lumen; extracellular region; plasma membrane Molecular Function:identical protein binding; protein binding; hormone activity; glucagon receptor binding; receptor binding Biological Process: positive regulation of protein binding; positive regulation of histone H3-K4 methylation; signal transduction; positive regulation of peptidyl-serine phosphorylation; G-protein signaling, coupled to cAMP nucleotide second messenger; response to starvation; G-protein coupled receptor protein signaling pathway; cell proliferation; cellular protein metabolic process; positive regulation of protein kinase activity; positive regulation of cAMP biosynthetic process; energy reserve metabolic process; feeding behavior; regulation of insulin secretion; negative regulation of apoptosis
NCBI Summary:	The protein encoded by this gene is actually a preproprotein that is cleaved into four distinct mature peptides. One of these, glucagon, is a pancreatic hormone that counteracts the glucose-lowering action of insulin by stimulating glycogenolysis and gluconeogenesis. Glucagon is a ligand for a specific G-protein linked receptor whose signalling pathway controls cell proliferation. Two of the other peptides are secreted from gut endocrine cells and promote nutrient absorption through distinct mechanisms. Finally, the fourth peptide is similar to glicentin, an active enteroglucagon. [provided by RefSeq, Jul 2008]
UniProt Code:	P01275
NCBI GenInfo Identifier:	125987831
NCBI Gene ID:	2641
NCBI Accession:	P01275.3
UniProt Secondary Accession:	P01275,Q53TP6, A6NN65,
UniProt Related Accession:	P01275
Molecular Weight:	20,909 Da
NCBI Full Name:	Glucagon
NCBI Synonym Full Names:	glucagon
NCBI Official Symbol:	GCG
NCBI Official Synonym Symbols:	GLP1; GLP2; GRPP
NCBI Protein Information:	glucagon; preproglucagon; glucagon-like peptide 1; glucagon-like peptide 2; glicentin-related polypeptide
UniProt Protein Name:	Glucagon
UniProt Synonym Protein Names:	Incretin hormone
Protein Family:	Glucagon
UniProt Gene Name:	GCG
UniProt Entry Name:	GLUC_HUMAN

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 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.

Step	Procedure
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-Glucagon 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.)