CRYAB (Phospho-Ser45)Colorimetric Cell-Based ELISA Kit (CBCAB01413)

Product Name:	CRYAB (Phospho-Ser45)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB01413
ELISA Type:	Cell-Based
Target:	CRYAB (Phospho-Ser45)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The CRYAB (Phospho-Ser45) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CRYAB protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated CRYAB in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on CRYAB phosphorylation.

Qualitative determination of CRYAB (Phospho-Ser45) concentration is achieved by an indirect ELISA format. In essence, CRYAB (Phospho-Ser45) is captured by CRYAB (Phospho-Ser45)-specific primary (1Ãƒ¸) antibodies while the HRP-conjugated secondary (2Ãƒ¸) antibodies bind the Fc region of the 1Ãƒ¸ antibody. Through this binding, the HRP enzyme conjugated to the 2Ãƒ¸ 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: 1410, UniProt ID: P02511, OMIM: 123590/608810, Unigene: Hs.408767
Gene Symbol:	CRYAB
Sub Type:	Phospho

UniProt Protein Function:	CRYAB: a major structural protein of the eye lens. A member of the small heat shock protein (sHSP also known as the HSP20) family. Alpha-B is expressed in the lens as well as other tissues. Elevated expression of alpha-B crystallin occurs in many neurological diseases; a missense mutation cosegregated in a family with a desmin-related myopathy.
UniProt Protein Details:	Protein type:Heat shock protein; Chaperone Chromosomal Location of Human Ortholog: 11q22.3-q23.1 Cellular Component: actin filament bundle; cell surface; cytoplasm; cytosol; Golgi apparatus; microtubule cytoskeleton; mitochondrion; myelin sheath; nucleoplasm; nucleus; plasma membrane; Z disc Molecular Function:identical protein binding; metal ion binding; microtubule binding; protein binding; protein homodimerization activity; structural constituent of eye lens; unfolded protein binding Biological Process: aging; lens development in camera-type eye; microtubule polymerization or depolymerization; muscle contraction; muscle development; negative regulation of apoptosis; negative regulation of caspase activity; negative regulation of cell growth; negative regulation of intracellular transport; protein folding; protein homooligomerization; response to estradiol stimulus; response to hydrogen peroxide; response to hypoxia; stress-activated MAPK cascade; tubulin folding Disease: Cardiomyopathy, Dilated, 1ii; Cataract 16, Multiple Types; Myopathy, Myofibrillar, 2; Myopathy, Myofibrillar, Fatal Infantile Hypertonic, Alpha-b Crystallin-related
NCBI Summary:	Mammalian lens crystallins are divided into alpha, beta, and gamma families. Alpha crystallins are composed of two gene products: alpha-A and alpha-B, for acidic and basic, respectively. Alpha crystallins can be induced by heat shock and are members of the small heat shock protein (HSP20) family. They act as molecular chaperones although they do not renature proteins and release them in the fashion of a true chaperone; instead they hold them in large soluble aggregates. Post-translational modifications decrease the ability to chaperone. These heterogeneous aggregates consist of 30-40 subunits; the alpha-A and alpha-B subunits have a 3:1 ratio, respectively. Two additional functions of alpha crystallins are an autokinase activity and participation in the intracellular architecture. The encoded protein has been identified as a moonlighting protein based on its ability to perform mechanistically distinct functions. Alpha-A and alpha-B gene products are differentially expressed; alpha-A is preferentially restricted to the lens and alpha-B is expressed widely in many tissues and organs. Elevated expression of alpha-B crystallin occurs in many neurological diseases; a missense mutation cosegregated in a family with a desmin-related myopathy. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jan 2014]
UniProt Code:	P02511
NCBI GenInfo Identifier:	117385
NCBI Gene ID:	1410
NCBI Accession:	P02511.2
UniProt Secondary Accession:	P02511,O43416, Q9UC37, Q9UC38, Q9UC39, Q9UC40, Q9UC41 B0YIX0,
UniProt Related Accession:	P02511
Molecular Weight:	20,159 Da
NCBI Full Name:	Alpha-crystallin B chain
NCBI Synonym Full Names:	crystallin alpha B
NCBI Official Symbol:	CRYAB
NCBI Official Synonym Symbols:	MFM2; CRYA2; CTPP2; HSPB5; CMD1II; CTRCT16; HEL-S-101
NCBI Protein Information:	alpha-crystallin B chain
UniProt Protein Name:	Alpha-crystallin B chain
UniProt Synonym Protein Names:	Alpha(B)-crystallin; Heat shock protein beta-5; HspB5; Renal carcinoma antigen NY-REN-27; Rosenthal fiber component
Protein Family:	Alpha-crystallin
UniProt Gene Name:	CRYAB
UniProt Entry Name:	CRYAB_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-CRYAB (Phospho-Ser45) Antibody, Anti-CRYAB 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.)