DGKA Colorimetric Cell-Based ELISA

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SKU:
CBCAB00991
€499

Description

Technical Manual

DGKA Cell-Based ELISA Kit

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

How does our DGKA Colorimetric Cell-Based ELISA Kit work?

Qualitative determination of DGKA concentration is achieved by an indirect ELISA format. In essence, DGKA is captured by DGKA -specific primary (1°) antibodies while the HRP-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the HRP enzyme conjugated to the 2° 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.

DGKA Colorimetric Cell-Based ELISA - Information

Product Name:DGKA Colorimetric Cell-Based ELISA
Product Code:/SKUCBCAB00991
Description:The DGKA Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect DGKA protein expression profile in cells. The kit can be used for measuring the relative amounts of DGKA in cultured cells as well as screening for the effects that various treatments, inhibitors (ie siRNA or chemicals), or activators have on DGKA.
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nm
Storage/Stability:4°C/6 Months
Reactivity:Human
Assay Type:Cell-Based
Database Links:Gene ID: 1606, UniProt ID: P23743, OMIM #: 125855, Unigene #: Hs.524488
Format:96-Well Microplate
NCBI Gene Symbol:DGKA
Sub Type:None
Target Name:DGKA

Kit Principle

Figure: Schematic representation of ELISA Genie Cell-Based Colorimetric ELISA principle

Kit components

Quantity

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 ul
100x Anti-Target Antibody60 ul
Anti-GAPDH Antibody60 ul
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

Additional equipment and materials required

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

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

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-DGKA 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.)

DGKA - Protein Information

UniProt Protein Function:DGKA: Upon cell stimulation converts the second messenger diacylglycerol into phosphatidate, initiating the resynthesis of phosphatidylinositols and attenuating protein kinase C activity. Monomer. Lymphocytes and oligodendroglial cells. Stimulated by calcium and phosphatidylserine. Phosphorylated by protein kinase C. Belongs to the eukaryotic diacylglycerol kinase family. 2 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:

Protein type:Lipid Metabolism - glycerophospholipid; EC 2.7.1.107; Motility/polarity/chemotaxis; Lipid Metabolism - glycerolipid; Kinase, lipid

Chromosomal Location of Human Ortholog: 12q13.3

Cellular Component: membrane; plasma membrane; cytosol

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

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

NCBI Summary:The protein encoded by this gene belongs to the eukaryotic diacylglycerol kinase family. It acts as a modulator that competes with protein kinase C for the second messenger diacylglycerol in intracellular signaling pathways. It also plays an important role in the resynthesis of phosphatidylinositols and phosphorylating diacylglycerol to phosphatidic acid. Alternative splicing occurs at this locus and four transcript variants encoding the same protein have been identified. [provided by RefSeq, Jul 2008]
UniProt Code:P23743
NCBI GenInfo Identifier:281185505
NCBI Gene ID:1606
NCBI Accession:P23743.3
UniProt Secondary Accession:P23743,O75481, O75482, O75483, O95217, Q3ZE25, Q8IZ56 Q8N5Q2,
UniProt Related Accession:P23743
Molecular Weight:Calculated: 13kDa/28kDa/82kDaObserved: 83kDa
NCBI Full Name:Diacylglycerol kinase alpha
NCBI Synonym Full Names:diacylglycerol kinase, alpha 80kDa
NCBI Official Symbol:DGKA  
NCBI Official Synonym Symbols:DAGK; DAGK1; DGK-alpha  
NCBI Protein Information:diacylglycerol kinase alpha; DAG kinase alpha; diglyceride kinase alpha; 80 kDa diacylglycerol kinase
UniProt Protein Name:Diacylglycerol kinase alpha
UniProt Synonym Protein Names:80 kDa diacylglycerol kinase; Diglyceride kinase alpha; DGK-alpha
Protein Family:Diacylglycerol kinase
UniProt Gene Name:DGKA  
UniProt Entry Name:DGKA_HUMAN
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