CKMT2 Colorimetric Cell-Based ELISA (CBCAB00978)

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Description

CKMT2 Colorimetric Cell-Based ELISA

The CKMT2 Colorimetric Cell-Based ELISA Kit is a powerful tool for detecting and quantifying CKMT2 levels in cell lysates and tissue extracts. This kit offers high sensitivity and accurate results, making it ideal for research in areas such as mitochondrial dysfunction, muscle diseases, and the role of CKMT2 in various cellular processes.CKMT2, also known as mitochondrial creatine kinase, is a key enzyme involved in energy metabolism within the mitochondria. It plays a crucial role in the generation of ATP, the energy currency of the cell, making it essential for proper muscle function and overall cellular health.

Dysregulation of CKMT2 has been linked to a variety of diseases, making it an important target for study and potential therapeutic interventions.With its user-friendly protocol and robust performance, the CKMT2 Colorimetric Cell-Based ELISA Kit is a valuable tool for researchers looking to explore the role of CKMT2 in health and disease. Obtain reliable and reproducible results with this innovative kit from Assay Genie.

Product Name:	CKMT2 Colorimetric Cell-Based ELISA
Product Code:	CBCAB00978
ELISA Type:	Cell-Based
Target:	CKMT2
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

The CKMT2 Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect CKMT2 protein expression profile in cells. The kit can be used for measuring the relative amounts of CKMT2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on CKMT2.

Qualitative determination of CKMT2 concentration is achieved by an indirect ELISA format. In essence, CKMT2 is captured by CKMT2-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: 1160, UniProt ID: P17540, OMIM: 123295, Unigene: Hs.80691
Gene Symbol:	CKMT2
Sub Type:	None

UniProt Protein Function:	CKMT2: Reversibly catalyzes the transfer of phosphate between ATP and various phosphogens (e.g. creatine phosphate). Creatine kinase isoenzymes play a central role in energy transduction in tissues with large, fluctuating energy demands, such as skeletal muscle, heart, brain and spermatozoa. Belongs to the ATP:guanido phosphotransferase family.
UniProt Protein Details:	Protein type:Mitochondrial; Amino Acid Metabolism - arginine and proline; Kinase, other; EC 2.7.3.2 Chromosomal Location of Human Ortholog: 5q13.3 Cellular Component: mitochondrial inner membrane; mitochondrion Molecular Function:creatine kinase activity Biological Process: creatine metabolic process; muscle contraction
NCBI Summary:	Mitochondrial creatine kinase (MtCK) is responsible for the transfer of high energy phosphate from mitochondria to the cytosolic carrier, creatine. It belongs to the creatine kinase isoenzyme family. It exists as two isoenzymes, sarcomeric MtCK and ubiquitous MtCK, encoded by separate genes. Mitochondrial creatine kinase occurs in two different oligomeric forms: dimers and octamers, in contrast to the exclusively dimeric cytosolic creatine kinase isoenzymes. Sarcomeric mitochondrial creatine kinase has 80% homology with the coding exons of ubiquitous mitochondrial creatine kinase. This gene contains sequences homologous to several motifs that are shared among some nuclear genes encoding mitochondrial proteins and thus may be essential for the coordinated activation of these genes during mitochondrial biogenesis. Three transcript variants encoding the same protein have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P17540
NCBI GenInfo Identifier:	116242603
NCBI Gene ID:	1160
NCBI Accession:	P17540.2
UniProt Secondary Accession:	P17540,Q6ICS8, Q8N1E1,
UniProt Related Accession:	P17540
Molecular Weight:	47,504 Da
NCBI Full Name:	Creatine kinase S-type, mitochondrial
NCBI Synonym Full Names:	creatine kinase, mitochondrial 2
NCBI Official Symbol:	CKMT2
NCBI Official Synonym Symbols:	SMTCK
NCBI Protein Information:	creatine kinase S-type, mitochondrial
UniProt Protein Name:	Creatine kinase S-type, mitochondrial
UniProt Synonym Protein Names:	Basic-type mitochondrial creatine kinase; Mib-CK; Sarcomeric mitochondrial creatine kinase; S-MtCK
Protein Family:	Creatine kinase
UniProt Gene Name:	CKMT2
UniProt Entry Name:	KCRS_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-CKMT2 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.)