SGK (Phospho-Ser422) Colorimetric Cell-Based ELISA Kit

Product Name:	SGK (Phospho-Ser422)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00403
ELISA Type:	Cell-Based
Target:	SGK (Phospho-Ser422)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The SGK (Phospho-Ser422) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect SGK protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated SGK in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on SGK phosphorylation.

Qualitative determination of SGK (Phospho-Ser422) concentration is achieved by an indirect ELISA format. In essence, SGK (Phospho-Ser422) is captured by SGK (Phospho-Ser422)-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: 6446, UniProt ID: O00141, OMIM: 602958, Unigene: Hs.510078
Gene Symbol:	SGK1
Sub Type:	Phospho

UniProt Protein Function:	SGK1: serum and glucocorticoid-inducible kinase is an AGC protein kinase of the SGK family. Rapidly induced in response to a variety of stimuli including serum, glucocorticoids, follicle stimulating hormone, osmotic shock and mineralocorticoids. Can be activated via PI3K-dependent and -independent pathways. Plays a role in activating certain potassium, sodium and chloride channels, suggesting an involvement in the regulation of processes such as cell survival, neuronal excitability and renal sodium excretion. SGK is negatively regulated by ubiquitin modification and proteasome degradation.
UniProt Protein Details:	Protein type:Protein kinase, AGC; Protein kinase, Ser/Thr (non-receptor); EC 2.7.11.1; Motility/polarity/chemotaxis; Kinase, protein; AGC group; SGK family Chromosomal Location of Human Ortholog: 6q23 Cellular Component: cytoplasm; cytosol; endoplasmic reticulum membrane; mitochondrion; neuron projection; nucleoplasm; nucleus; perinuclear region of cytoplasm; plasma membrane Molecular Function:3-phosphoinositide-dependent protein kinase binding; ATP binding; calcium channel regulator activity; chloride channel regulator activity; cofactor binding; potassium channel regulator activity; protein binding; protein serine/threonine kinase activity; protein serine/threonine/tyrosine kinase activity; sodium channel regulator activity; tau protein binding Biological Process: apoptosis; cellular sodium ion homeostasis; glucocorticoid mediated signaling; long-term memory; microtubule depolymerization; negative regulation of apoptosis; negative regulation of microtubule polymerization; neurite morphogenesis; peptidyl-serine phosphorylation; positive regulation of cell growth; positive regulation of dendrite morphogenesis; positive regulation of transporter activity; protein amino acid phosphorylation; regulation of apoptosis; regulation of blood pressure; regulation of catalytic activity; regulation of cell cycle; regulation of cell growth; regulation of cell migration; regulation of cell proliferation; regulation of transcription factor activity; response to DNA damage stimulus; response to stress; sodium ion transport; transmembrane transport; visual learning
NCBI Summary:	This gene encodes a serine/threonine protein kinase that plays an important role in cellular stress response. This kinase activates certain potassium, sodium, and chloride channels, suggesting an involvement in the regulation of processes such as cell survival, neuronal excitability, and renal sodium excretion. High levels of expression of this gene may contribute to conditions such as hypertension and diabetic nephropathy. Several alternatively spliced transcript variants encoding different isoforms have been noted for this gene. [provided by RefSeq, Jan 2009]
UniProt Code:	O00141
NCBI GenInfo Identifier:	90185131
NCBI Gene ID:	6446
NCBI Accession:	O00141.2
UniProt Secondary Accession:	O00141,Q5TCN2, Q5TCN3, Q5TCN4, Q5VY65, Q9UN56, B7UUP7 B7UUP8, B7UUP9, B7Z5B2, E1P583,
UniProt Related Accession:	O00141
Molecular Weight:
NCBI Full Name:	Serine/threonine-protein kinase Sgk1
NCBI Synonym Full Names:	serum/glucocorticoid regulated kinase 1
NCBI Official Symbol:	SGK1
NCBI Official Synonym Symbols:	SGK
NCBI Protein Information:	serine/threonine-protein kinase Sgk1
UniProt Protein Name:	Serine/threonine-protein kinase Sgk1
UniProt Synonym Protein Names:	Serum/glucocorticoid-regulated kinase 1
Protein Family:	Serine/threonine-protein kinase
UniProt Gene Name:	SGK1
UniProt Entry Name:	SGK1_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-SGK (Phospho-Ser422) Antibody, Anti-SGK 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.)