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PKC epsilon Colorimetric Cell-Based ELISA Kit

SKU:
CBCAB00817
Product Type:
ELISA Kit
ELISA Type:
Cell Based
Research Area:
Cell Biology
Reactivity:
Human
Mouse
Rat
Detection Method:
Colorimetric
$719
Frequently bought together:

Description

system_update_altDatasheetMSDS

PKC epsilon Colorimetric Cell-Based ELISA Kit

The PKC Epsilon Colorimetric Cell-Based ELISA Kit is a reliable and accurate tool for detecting and quantifying the levels of PKC Epsilon in cell lysates and tissue homogenates. This kit offers high sensitivity and specificity, ensuring precise and reproducible results for a variety of research applications.PKC Epsilon is an important protein kinase C isoform that plays a critical role in regulating cell proliferation, differentiation, and apoptosis. Dysregulation of PKC Epsilon has been linked to various diseases, including cancer, diabetes, and neurological disorders.

Therefore, measuring PKC Epsilon levels can provide valuable insights into disease mechanisms and potential therapeutic targets.With easy-to-follow protocols and rapid assay times, the PKC Epsilon Colorimetric Cell-Based ELISA Kit is a valuable tool for researchers aiming to understand the role of PKC Epsilon in cellular signaling pathways and disease pathogenesis. Invest in this kit today to advance your research efforts in the field of cell biology and disease biology.

Product Name:PKC epsilon Colorimetric Cell-Based ELISA
Product Code:CBCAB00817
ELISA Type:Cell-Based
Target:PKC epsilon
Reactivity:Human, Mouse, Rat
Dynamic Range:> 5000 Cells
Detection Method:Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:96-Well Microplate

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

Qualitative determination of PKC epsilon concentration is achieved by an indirect ELISA format. In essence, PKC epsilon is captured by PKC epsilon-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: 5581, UniProt ID: Q02156, OMIM: 176975, Unigene: Hs.580351
Gene Symbol:PRKCE
Sub Type:None
UniProt Protein Function:PKCE: an AGC kinase of the PKC family. Calcium-independent, phospholipid-dependent. Activated by inflammatory mediators and involved in nociceptive functions. PKC-epsilon null mice display decreased hypoxic pulmonary vasoconstriction.
UniProt Protein Details:

Protein type:Kinase, protein; Protein kinase, AGC; EC 2.7.11.13; Protein kinase, Ser/Thr (non-receptor); AGC group; PKC family; Eta subfamily

Chromosomal Location of Human Ortholog: 2p21

Cellular Component: cytoplasm; cytosol; endoplasmic reticulum; Golgi apparatus; perinuclear region of cytoplasm; plasma membrane

Molecular Function:actin monomer binding; enzyme activator activity; enzyme binding; protein binding; protein kinase C activity; protein serine/threonine kinase activity; signal transducer activity

Biological Process: apoptosis; lipopolysaccharide-mediated signaling pathway; peptidyl-serine phosphorylation; phospholipase C activation; platelet activation; positive regulation of actin filament polymerization; positive regulation of cytokinesis; protein amino acid phosphorylation; signal transduction

NCBI Summary:Protein kinase C (PKC) is a family of serine- and threonine-specific protein kinases that can be activated by calcium and the second messenger diacylglycerol. PKC family members phosphorylate a wide variety of protein targets and are known to be involved in diverse cellular signaling pathways. PKC family members also serve as major receptors for phorbol esters, a class of tumor promoters. Each member of the PKC family has a specific expression profile and is believed to play a distinct role in cells. The protein encoded by this gene is one of the PKC family members. This kinase has been shown to be involved in many different cellular functions, such as neuron channel activation, apoptosis, cardioprotection from ischemia, heat shock response, as well as insulin exocytosis. Knockout studies in mice suggest that this kinase is important for lipopolysaccharide (LPS)-mediated signaling in activated macrophages and may also play a role in controlling anxiety-like behavior. [provided by RefSeq, Jul 2008]
UniProt Code:Q02156
NCBI GenInfo Identifier:400135
NCBI Gene ID:5581
NCBI Accession:Q02156.1
UniProt Secondary Accession:Q02156,Q32MQ3, Q53SL4, Q53SM5, Q9UE81, B0LPH7,
UniProt Related Accession:Q02156
Molecular Weight:83,674 Da
NCBI Full Name:Protein kinase C epsilon type
NCBI Synonym Full Names:protein kinase C epsilon
NCBI Official Symbol:PRKCE  
NCBI Official Synonym Symbols:PKCE; nPKC-epsilon  
NCBI Protein Information:protein kinase C epsilon type
UniProt Protein Name:Protein kinase C epsilon type
UniProt Synonym Protein Names:nPKC-epsilon
Protein Family:Protein kinase
UniProt Gene Name:PRKCE  
UniProt Entry Name:KPCE_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-PKC epsilon 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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