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DGKE Colorimetric Cell-Based ELISA

SKU:
CBCAB01031
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheet

DGKE Colorimetric Cell-Based ELISA

The DGKE Colorimetric Cell-Based ELISA Kit is a cutting-edge tool for the precise measurement of diacylglycerol kinase epsilon (DGKE) levels in cell lysates and tissue samples. This kit offers superior sensitivity and accuracy, ensuring robust and consistent results for a variety of research purposes.DGKE is a critical enzyme involved in the regulation of lipid metabolism and signaling pathways, making it a key player in various cellular processes. Dysregulation of DGKE has been linked to metabolic disorders, cancer, and neurological conditions, highlighting its importance as a potential therapeutic target.

With the DGKE Colorimetric Cell-Based ELISA Kit, researchers can accurately quantify DGKE levels in their samples, allowing for in-depth exploration of its functions and potential implications in disease pathology. This powerful kit is a valuable asset for any laboratory aiming to unravel the intricate mechanisms underlying cellular function and disease development.

Product Name:DGKE Colorimetric Cell-Based ELISA
Product Code:CBCAB01031
ELISA Type:Cell-Based
Target:DGKE
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of DGKE concentration is achieved by an indirect ELISA format. In essence, DGKE is captured by DGKE-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: 8526, UniProt ID: P52429, OMIM: 601440, Unigene: Hs.239514
Gene Symbol:DGKE
Sub Type:None
UniProt Protein Function:DGKE: Highly selective for arachidonate-containing species of diacylglycerol (DAG). May terminate signals transmitted through arachidonoyl-DAG or may contribute to the synthesis of phospholipids with defined fatty acid composition. Belongs to the eukaryotic diacylglycerol kinase family.
UniProt Protein Details:

Protein type:Lipid Metabolism - glycerolipid; Lipid Metabolism - glycerophospholipid; Membrane protein, multi-pass; Membrane protein, integral; EC 2.7.1.107; Motility/polarity/chemotaxis; Kinase, lipid

Chromosomal Location of Human Ortholog: 17q22

Cellular Component: membrane; cytoplasm; integral to membrane; plasma membrane

Molecular Function:metal ion binding; diacylglycerol kinase activity; ATP binding; NAD+ kinase activity

Biological Process: platelet activation; protein kinase C activation; blood coagulation; phosphorylation; phospholipid biosynthetic process

Disease: Nephrotic Syndrome, Type 7

NCBI Summary:Diacylglycerol kinases are thought to be involved mainly in the regeneration of phosphatidylinositol (PI) from diacylglycerol in the PI-cycle during cell signal transduction. When expressed in mammalian cells, DGK-epsilon shows specificity for arachidonyl-containing diacylglycerol. DGK-epsilon is expressed predominantly in testis. [provided by RefSeq, Jul 2008]
UniProt Code:P52429
NCBI GenInfo Identifier:4503313
NCBI Gene ID:8526
NCBI Accession:NP_003638.1
UniProt Related Accession:P52429
Molecular Weight:~ 64kDa
NCBI Full Name:diacylglycerol kinase epsilon
NCBI Synonym Full Names:diacylglycerol kinase epsilon
NCBI Official Symbol:DGKE  
NCBI Official Synonym Symbols:DGK; AHUS7; DAGK5; DAGK6; NPHS7  
NCBI Protein Information:diacylglycerol kinase epsilon
UniProt Protein Name:Diacylglycerol kinase epsilon
UniProt Synonym Protein Names:Diglyceride kinase epsilon
Protein Family:Deoxyguanosine kinase
UniProt Gene Name:DGKE  
UniProt Entry Name:DGKE_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-DGKE 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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