BLNK Colorimetric Cell-Based ELISA Kit (CBCAB00548)

Product Name:	BLNK Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00548
ELISA Type:	Cell-Based
Target:	BLNK
Reactivity:	Human, Mouse
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of BLNK concentration is achieved by an indirect ELISA format. In essence, BLNK is captured by BLNK-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: 29760, UniProt ID: Q8WV28, OMIM: 604515, Unigene: Hs.665244
Gene Symbol:	BLNK
Sub Type:	None

UniProt Protein Function:	BLNK: an adaptor protein that bridges the B-cell receptor-associated kinases (BCR) with a multitude of signaling pathways, regulating biologic outcomes of B-cell function and development. Plays an important role in BCR-mediated PLCG1 activation and Ca(2 ) mobilization. Does not seem to be not required for pre-BCR-mediated activation of MAP kinase and phosphatidyl-inositol 3 (PI3) kinase signaling. Plays a critical role in orchestrating the pro-B cell to pre-B cell transition. Following BCR activation, phosphorylated on tyrosine residues by SYK and LYN. When phosphorylated, serves as a scaffold to assemble downstream targets of antigen activation, including PLCG1, VAV1, GRB2 and NCK1. Its phosphorylation is required for both Ca(2 ) and MAPK signaling pathways. Defects in BLNK are the cause of hypoglobulinemia and absent B-cells.It has tumor supressor activity that is lost in many cases of childhood pre-B acute lymphoblastic leukemia (ALL). Two alternatively spliced isoforms have been described; these are differentially involved in activation and apoptosis of B lymphocytes.
UniProt Protein Details:	Protein type:Adaptor/scaffold Chromosomal Location of Human Ortholog: 10q23.2-q23.33 Cellular Component: cytoplasm; cytosol; plasma membrane Molecular Function:protein binding; SH3/SH2 adaptor activity; transmembrane receptor protein tyrosine kinase adaptor protein activity Biological Process: humoral immune response; inflammatory response Disease: Agammaglobulinemia 4, Autosomal Recessive
NCBI Summary:	This gene encodes a cytoplasmic linker or adaptor protein that plays a critical role in B cell development. This protein bridges B cell receptor-associated kinase activation with downstream signaling pathways, thereby affecting various biological functions. The phosphorylation of five tyrosine residues is necessary for this protein to nucleate distinct signaling effectors following B cell receptor activation. Mutations in this gene cause hypoglobulinemia and absent B cells, a disease in which the pro- to pre-B-cell transition is developmentally blocked. Deficiency in this protein has also been shown in some cases of pre-B acute lymphoblastic leukemia. Alternatively spliced transcript variants have been found for this gene. [provided by RefSeq, May 2012]
UniProt Code:	Q8WV28
NCBI GenInfo Identifier:	82592659
NCBI Gene ID:	29760
NCBI Accession:	Q8WV28.2
UniProt Secondary Accession:	Q8WV28,O75498, O75499, Q2MD49,
UniProt Related Accession:	Q8WV28
Molecular Weight:	44,451 Da
NCBI Full Name:	B-cell linker protein
NCBI Synonym Full Names:	B-cell linker
NCBI Official Symbol:	BLNK
NCBI Official Synonym Symbols:	bca; AGM4; BASH; LY57; SLP65; BLNK-S; SLP-65
NCBI Protein Information:	B-cell linker protein
UniProt Protein Name:	B-cell linker protein
UniProt Synonym Protein Names:	B-cell adapter containing a SH2 domain protein; B-cell adapter containing a Src homology 2 domain protein; Cytoplasmic adapter protein; Src homology 2 domain-containing leukocyte protein of 65 kDa; SLP-65
Protein Family:	B-cell linker protein
UniProt Gene Name:	BLNK
UniProt Entry Name:	BLNK_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-BLNK 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.)