GSK3alpha (Phospho-Ser21)Colorimetric Cell-Based ELISA Kit (CBCAB01537)

Product Name:	GSK3alpha (Phospho-Ser21)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01537
ELISA Type:	Cell-Based
Target:	GSK3alpha (Phospho-Ser21)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The GSK3alpha (Phospho-Ser21) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect GSK3alpha protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated GSK3alpha in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on GSK3alpha phosphorylation.

Qualitative determination of GSK3alpha (Phospho-Ser21) concentration is achieved by an indirect ELISA format. In essence, GSK3alpha (Phospho-Ser21) is captured by GSK3alpha (Phospho-Ser21)-specific primary (1Ã¸) antibodies while the HRP-conjugated secondary (2Ã¸) antibodies bind the Fc region of the 1Ã¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ã¸ 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: 2931, UniProt ID: P49840, OMIM: 606784, Unigene: Hs.466829
Gene Symbol:	GSK3A
Sub Type:	Phospho

UniProt Protein Function:	GSK3A: a proline-directed protein kinase of the GSK family. Implicated in the control of several regulatory proteins including glycogen synthase, Myb, and c-Jun. GSK3 and GSK3 have similar functions. GSK3 phophorylates tau, the principal component of neurofibrillary tangles in Alzheimer disease and is required for maximal production of amyloid plaque peptides by secretase. A GSK3 promoter SNP effects progression of bipolar disorder. The GSK3 inhibitor, lithium, is used to treat bipolar disorder and is seen to block plaque formation. GSK3 generally opposes the action of insulin, and GSK3 hyperactivity is thought to contribute to insulin resistant (type II) diabetes. GSK3 also negatively regulates cardiac hypertrophy. A tumor suppressor role is indicated by the oncogenic potential of stabilized -catenin mutants that lack GSK3 phosphorylation sites. Inhibitor: AR-A014418
UniProt Protein Details:	Protein type:EC 2.7.11.26; EC 2.7.11.1; Protein kinase, CMGC; Kinase, protein; Protein kinase, Ser/Thr (non-receptor); CMGC group; GSK family; GSK subfamily Chromosomal Location of Human Ortholog: 19q13.2 Cellular Component: beta-catenin destruction complex; cytosol Molecular Function:protein binding; protein serine/threonine kinase activity; tau-protein kinase activity Biological Process: cellular response to insulin stimulus; insulin receptor signaling pathway; negative regulation of glucose import; negative regulation of glycogen biosynthetic process; negative regulation of insulin receptor signaling pathway; negative regulation of TOR signaling pathway; positive regulation of cAMP biosynthetic process; positive regulation of heart contraction; proteasomal ubiquitin-dependent protein catabolic process; protein amino acid phosphorylation; regulation of systemic arterial blood pressure
NCBI Summary:	This gene encodes a multifunctional Ser/Thr protein kinase that is implicated in the control of several regulatory proteins including glycogen synthase, and transcription factors, such as JUN. It also plays a role in the WNT and PI3K signaling pathways, as well as regulates the production of beta-amyloid peptides associated with Alzheimer's disease. [provided by RefSeq, Oct 2011]
UniProt Code:	P49840
NCBI GenInfo Identifier:	12644292
NCBI Gene ID:	2931
NCBI Accession:	P49840.2
UniProt Secondary Accession:	P49840,O14959,
UniProt Related Accession:	P49840
Molecular Weight:	50,981 Da
NCBI Full Name:	Glycogen synthase kinase-3 alpha
NCBI Synonym Full Names:	glycogen synthase kinase 3 alpha
NCBI Official Symbol:	GSK3A
NCBI Protein Information:	glycogen synthase kinase-3 alpha
UniProt Protein Name:	Glycogen synthase kinase-3 alpha
UniProt Synonym Protein Names:	Serine/threonine-protein kinase GSK3A (EC:2.7.11.1)
Protein Family:	Glycogen synthase kinase
UniProt Gene Name:	GSK3A
UniProt Entry Name:	GSK3A_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-GSK3alpha (Phospho-Ser21) Antibody, Anti-GSK3alpha 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.)