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DGKI Colorimetric Cell-Based ELISA

SKU:
CBCAB00957
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Reactivity:
Human
Rat
Detection Method:
Colorimetric
€599
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Description

system_update_altDatasheetMSDS

DGKI Colorimetric Cell-Based ELISA

The DGK1 Colorimetric Cell-Based ELISA Kit is specifically designed for the quantitative detection of DGK1 levels in cell lysates. This kit offers high sensitivity and accuracy, providing researchers with reliable and reproducible results.DGK1, also known as Diacylglycerol Kinase 1, is a key enzyme involved in lipid metabolism and signal transduction pathways. It plays a critical role in regulating cell growth, differentiation, and apoptosis. Dysregulation of DGK1 has been linked to various diseases, including cancer, autoimmune disorders, and neurological conditions, highlighting its potential as a therapeutic target.

With the DGK1 Colorimetric Cell-Based ELISA Kit, researchers can gain valuable insights into the role of DGK1 in cellular processes and disease development. This kit is suitable for a wide range of experimental settings, making it a versatile tool for advancing biomedical research.

Product Name:DGKI Colorimetric Cell-Based ELISA
Product Code:CBCAB00957
ELISA Type:Cell-Based
Target:DGKI
Reactivity:Human, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of DGKI concentration is achieved by an indirect ELISA format. In essence, DGKI is captured by DGKI-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: 9162, UniProt ID: O75912, OMIM: 604072, Unigene: Hs.242947
Gene Symbol:DGKI
Sub Type:None
UniProt Protein Function:DGKI: a member of the type IV diacylglycerol kinase subfamily. Diacylglycerol kinases regulate the intracellular concentration of diacylglycerol through its phosphorylation, producing phosphatidic acid. The specific role of the enzyme encoded by undetermined, however, it may play a crucial role in the production of phosphatidic acid in the retina or in recessive forms of retinal degeneration. [provided by RefSeq, Jul 2008]
UniProt Protein Details:

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

Chromosomal Location of Human Ortholog: 7q32.3-q33

Cellular Component: cytoplasm; dendritic spine; nucleoplasm; nucleus; plasma membrane; protein complex; synapse

Molecular Function:diacylglycerol kinase activity; protein binding

Biological Process: platelet activation

NCBI Summary:This gene is a member of the type IV diacylglycerol kinase subfamily. Diacylglycerol kinases regulate the intracellular concentration of diacylglycerol through its phosphorylation, producing phosphatidic acid. The specific role of the enzyme encoded by this gene is undetermined, however, it may play a crucial role in the production of phosphatidic acid in the retina or in recessive forms of retinal degeneration. [provided by RefSeq, Jul 2008]
UniProt Code:O75912
NCBI GenInfo Identifier:12643642
NCBI Gene ID:9162
NCBI Accession:O75912.1
UniProt Secondary Accession:O75912,Q9NZ49, A4D1Q9,
UniProt Related Accession:O75912
Molecular Weight:116,997 Da
NCBI Full Name:Diacylglycerol kinase iota
NCBI Synonym Full Names:diacylglycerol kinase iota
NCBI Official Symbol:DGKI  
NCBI Official Synonym Symbols:DGK-IOTA  
NCBI Protein Information:diacylglycerol kinase iota
UniProt Protein Name:Diacylglycerol kinase iota
UniProt Synonym Protein Names:Diglyceride kinase iota; DGK-iota
Protein Family:Diacylglycerol kinase
UniProt Gene Name:DGKI  
UniProt Entry Name:DGKI_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-DGKI 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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