LKB1 (Phospho-Ser428) Colorimetric Cell-Based ELISA Kit

Product Name:	LKB1 (Phospho-Ser428) Colorimetric Cell-Based ELISA
Product Code:	CBCAB00046
ELISA Type:	Cell-Based
Target:	LKB1 (Phospho-Ser428)
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The LKB1 (Phospho-Ser428) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect LKB1 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated LKB1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on LKB1 phosphorylation.

Qualitative determination of LKB1 (Phospho-Ser428) concentration is achieved by an indirect ELISA format. In essence, LKB1 (Phospho-Ser428) is captured by LKB1 (Phospho-Ser428)-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: 6794, UniProt ID: Q15831, OMIM: 175200/273300/602216, Unigene: Hs.515005
Gene Symbol:	STK11
Sub Type:	Phospho

UniProt Protein Function:	LKB1: a S/T protein kinase of the CAMKL family. A tumor suppressor that helps control cell structure, polarity, apoptosis and energy homeostasis. Phosphorylates AGS3 (activator of G-protein signaling 3) GPR domains, regulating the interaction of GPR-containing proteins with G-proteins. A cytosolic protein complex comprised of LKB1, the pseudokinase STRAD, and the MO25 scaffold protein. Activates AMPK and several related protein kinases. AMPK plays a predominant role as the master regulator of cellular energy homeostasis, controlling downstream effectors that regulate cell growth and apoptosis in response to cellular ATP concentrations. The Lkb1/Strad/Mo25 complex can polarize single epithelial cells, leading to the formation of a brush border containing microvilli on the apical surface. Mutation in the corresponding gene causes Peutz-Jeghers syndrome (PJS), an autosomal dominant disorder characterized by benign GI tract polyps and dark skin lesions of the mouth, hands and feet. A variety of other LKB1 gene mutations have been associated with the formation of sporadic cancers in several tissues.
UniProt Protein Details:	Protein type:Protein kinase, CAMK; Protein kinase, Ser/Thr (non-receptor); Tumor suppressor; Autophagy; EC 2.7.11.1; Kinase, protein; CAMK group; CAMKL family; LKB subfamily Chromosomal Location of Human Ortholog: 19p13.3 Cellular Component: cytoplasm; cytosol; membrane; mitochondrion; nucleus Molecular Function:ATP binding; LRR domain binding; magnesium ion binding; p53 binding; protein binding; protein kinase activator activity; protein serine/threonine kinase activity Biological Process: activation of protein kinase activity; anoikis; autophagy; axonogenesis; cell cycle arrest; energy reserve metabolic process; establishment of cell polarity; glucose homeostasis; Golgi localization; insulin receptor signaling pathway; negative regulation of cell growth; negative regulation of cell proliferation; positive regulation of autophagy; positive regulation of axonogenesis; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of transforming growth factor beta receptor signaling pathway; positive thymic T cell selection; protein amino acid autophosphorylation; protein amino acid phosphorylation; regulation of cell growth; regulation of dendrite morphogenesis; regulation of fatty acid biosynthetic process; regulation of protein kinase B signaling cascade; regulation of Wnt receptor signaling pathway; response to DNA damage stimulus; response to ionizing radiation; spermatid development; T cell receptor signaling pathway; tissue homeostasis; vasculature development; Wnt receptor signaling pathway through beta-catenin Disease: Pancreatic Cancer; Peutz-jeghers Syndrome; Testicular Germ Cell Tumor
NCBI Summary:	This gene, which encodes a member of the serine/threonine kinase family, regulates cell polarity and functions as a tumor suppressor. Mutations in this gene have been associated with Peutz-Jeghers syndrome, an autosomal dominant disorder characterized by the growth of polyps in the gastrointestinal tract, pigmented macules on the skin and mouth, and other neoplasms. Alternate transcriptional splice variants of this gene have been observed but have not been thoroughly characterized. [provided by RefSeq, Jul 2008]
UniProt Code:	Q15831
NCBI GenInfo Identifier:	3024670
NCBI Gene ID:	6794
NCBI Accession:	Q15831.1
UniProt Secondary Accession:	Q15831,B2RBX7, E7EW76,
UniProt Related Accession:	Q15831
Molecular Weight:	45,387 Da
NCBI Full Name:	Serine/threonine-protein kinase STK11
NCBI Synonym Full Names:	serine/threonine kinase 11
NCBI Official Symbol:	STK11
NCBI Official Synonym Symbols:	PJS; LKB1; hLKB1
NCBI Protein Information:	serine/threonine-protein kinase STK11
UniProt Protein Name:	Serine/threonine-protein kinase STK11
UniProt Synonym Protein Names:	Liver kinase B1; LKB1; hLKB1; Renal carcinoma antigen NY-REN-19
Protein Family:	Serine/threonine-protein kinase
UniProt Gene Name:	STK11
UniProt Entry Name:	STK11_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-LKB1 (Phospho-Ser428) Antibody, Anti-LKB1 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.)

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신경면역학: CNS의 면역체계

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LKB1 (Phospho-Ser428) Colorimetric Cell-Based ELISA Kit

Description