PBK/TOPK Colorimetric Cell-Based ELISA Kit (CBCAB00804)

Product Name:	PBK/TOPK Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00804
ELISA Type:	Cell-Based
Target:	PBK/TOPK
Reactivity:	Human
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of PBK/TOPK concentration is achieved by an indirect ELISA format. In essence, PBK/TOPK is captured by PBK/TOPK-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: 55872, UniProt ID: Q96KB5, OMIM: 611210, Unigene: Hs.104741
Gene Symbol:	PBK
Sub Type:	None

UniProt Protein Function:	PBK: Phosphorylates MAP kinase p38. Seems to be active only in mitosis. May also play a role in the activation of lymphoid cells. When phosphorylated, forms a complex with TP53, leading to TP53 destabilization and attenuation of G2/M checkpoint during doxorubicin-induced DNA damage. Interacts with DLG1 and TP53. Expressed in the testis and placenta. In the testis, restrictedly expressed in outer cell layer of seminiferous tubules. Activated by phosphorylation. Belongs to the protein kinase superfamily. Ser/Thr protein kinase family. MAP kinase kinase subfamily.
UniProt Protein Details:	Protein type:EC 2.7.12.2; Cancer Testis Antigen (CTA); Protein kinase, Ser/Thr (non-receptor); Kinase, protein; Protein kinase, Other; Other group; TOPK family Chromosomal Location of Human Ortholog: 8p21.2 Cellular Component: nucleus Molecular Function:ATP binding; protein binding; protein serine/threonine kinase activity Biological Process: mitosis; negative regulation of inflammatory response; negative regulation of proteasomal ubiquitin-dependent protein catabolic process; negative regulation of stress-activated MAPK cascade; protein amino acid phosphorylation
NCBI Summary:	This gene encodes a serine/threonine protein kinase related to the dual specific mitogen-activated protein kinase kinase (MAPKK) family. Evidence suggests that mitotic phosphorylation is required for its catalytic activity. The encoded protein may be involved in the activation of lymphoid cells and support testicular functions, with a suggested role in the process of spermatogenesis. Overexpression of this gene has been implicated in tumorigenesis. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Jul 2013]
UniProt Code:	Q96KB5
NCBI GenInfo Identifier:	83305809
NCBI Gene ID:	55872
NCBI Accession:	Q96KB5.3
UniProt Secondary Accession:	Q96KB5,Q9NPD9, Q9NYL7, Q9NZK6, B4DX68, D3DST2,
UniProt Related Accession:	Q96KB5
Molecular Weight:	37,211 Da
NCBI Full Name:	Lymphokine-activated killer T-cell-originated protein kinase
NCBI Synonym Full Names:	PDZ binding kinase
NCBI Official Symbol:	PBK
NCBI Official Synonym Symbols:	SPK; CT84; TOPK; HEL164; Nori-3
NCBI Protein Information:	lymphokine-activated killer T-cell-originated protein kinase
UniProt Protein Name:	Lymphokine-activated killer T-cell-originated protein kinase
UniProt Synonym Protein Names:	Cancer/testis antigen 84; CT84; MAPKK-like protein kinase; Nori-3; PDZ-binding kinase; Spermatogenesis-related protein kinase; SPK; T-LAK cell-originated protein kinase
Protein Family:	Lymphokine-activated killer T-cell-originated protein kinase
UniProt Gene Name:	PBK
UniProt Entry Name:	TOPK_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-PBK/TOPK 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.)