Metabolism

CKII-beta Colorimetric Cell-Based ELISA Kit

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SKU:
CBCAB00582
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Metabolism
Reactivity:
Human
Reactivity:
Mouse
Reactivity:
Rat
Detection Method:
Colorimetric

Description

system_update_altDatasheet
Product Name:CKII-beta Colorimetric Cell-Based ELISA
Product Code:CBCAB00582
ELISA Type:Cell-Based
Target:CKII-beta
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of CKII-beta concentration is achieved by an indirect ELISA format. In essence, CKII-beta is captured by CKII-beta-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: 1460, UniProt ID: P67870, OMIM: 115441, Unigene: Hs.73527
Gene Symbol:CSNK2B
Sub Type:None
UniProt Protein Function:CK2B: a regulatory subunit of the casein kinase 2 holoenzyme. Exists as a tetramer composed of two catalytic subunits, alpha and alpha-prime, and two regulatory beta subunits. The beta subunits undergo autophosphorylation. Plays a complex role in regulating the basal catalytic activity of the alpha subunit. Participates in Wnt signaling
UniProt Protein Details:

Protein type:Protein kinase, regulatory subunit

Chromosomal Location of Human Ortholog: 6p21.3

Cellular Component: cytoplasm; cytosol; nucleoplasm; nucleus; PcG protein complex; plasma membrane

Molecular Function:chromatin binding; identical protein binding; protein binding; protein domain specific binding; protein serine/threonine kinase activity; receptor binding; transcription factor binding

Biological Process: adiponectin-mediated signaling pathway; negative regulation of blood vessel endothelial cell migration; negative regulation of cell proliferation; positive regulation of activin receptor signaling pathway; protein folding; signal transduction

NCBI Summary:This gene encodes the beta subunit of casein kinase II, a ubiquitous protein kinase which regulates metabolic pathways, signal transduction, transcription, translation, and replication. The enzyme is composed of three subunits, alpha, alpha prime and beta, which form a tetrameric holoenzyme. The alpha and alpha prime subunits are catalytic, while the beta subunit serves regulatory functions. The enzyme localizes to the endoplasmic reticulum and the Golgi apparatus. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Sep 2013]
UniProt Code:P67870
NCBI GenInfo Identifier:54037520
NCBI Gene ID:1460
NCBI Accession:P67870.1
UniProt Secondary Accession:P67870,P07312, P13862, Q4VX47, B0UXA9,
UniProt Related Accession:P67870
Molecular Weight:24,942 Da
NCBI Full Name:Casein kinase II subunit beta
NCBI Synonym Full Names:casein kinase 2 beta
NCBI Official Symbol:CSNK2B  
NCBI Official Synonym Symbols:G5A; CK2B; CK2N; CSK2B  
NCBI Protein Information:casein kinase II subunit beta
UniProt Protein Name:Casein kinase II subunit beta
UniProt Synonym Protein Names:Phosvitin; Protein G5a
UniProt Gene Name:CSNK2B  
UniProt Entry Name:CSK2B_HUMAN
ComponentQuantity
96-Well Cell Culture Clear-Bottom Microplate2 plates
10X TBS24 mL
Quenching Buffer24 mL
Blocking Buffer50 mL
15X Wash Buffer50 mL
Primary Antibody Diluent12 mL
100x Anti-Phospho Target Antibody 60 µL
100x Anti-Target Antibody60 µL
Anti-GAPDH Antibody60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody12 mL
HRP-Conjugated Anti-Mouse IgG Antibody12 mL
SDS Solution12 mL
Stop Solution24 mL
Ready-to-Use Substrate12 mL
Crystal Violet Solution12 mL
Adhesive Plate Seals2 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.

StepProcedure
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-CKII-beta 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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