PKC theta Colorimetric Cell-Based ELISA Kit (CBCAB00819)

Product Name:	PKC theta Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00819
ELISA Type:	Cell-Based
Target:	PKC theta
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nmStorage/Stability:4°C/6 Months
Format:	96-Well Microplate

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

Qualitative determination of PKC theta concentration is achieved by an indirect ELISA format. In essence, PKC theta is captured by PKC theta-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: 5588, UniProt ID: Q04759, OMIM: 600448, Unigene: Hs.498570
Gene Symbol:	PRKCQ
Sub Type:	None

UniProt Protein Function:	PKCT: an AGC kinase of the PKC family. A novel PKC: Ca2+ independent but still regulated by PS, DAG and phorbol esters. Contains 2 zinc-dependent phorbol-ester and DAG binding domains. Expressed in skeletal muscle and hematopoietic cells. May play a role in T cell receptor signaling and insulin resistance. Required for TCR-induced NF-kappaB activation in mature T lymphocytes.
UniProt Protein Details:	Protein type:Kinase, protein; EC 2.7.11.13; Protein kinase, Ser/Thr (non-receptor); Protein kinase, AGC; AGC group; PKC family; Delta subfamily Chromosomal Location of Human Ortholog: 10p15 Cellular Component: plasma membrane; immunological synapse; cytosol Molecular Function:protein serine/threonine kinase activity; protein binding; protein kinase C activity; metal ion binding; ubiquitin-protein ligase activity; ATP binding Biological Process: positive regulation of interleukin-17 production; axon guidance; phototransduction, visible light; platelet activation; apoptosis; positive regulation of interleukin-2 biosynthetic process; membrane protein ectodomain proteolysis; negative regulation of insulin receptor signaling pathway; protein ubiquitination; protein amino acid phosphorylation; T cell receptor signaling pathway; activation of NF-kappaB transcription factor; rhodopsin mediated signaling; positive regulation of interleukin-4 production; regulation of rhodopsin mediated signaling; regulation of transcription, DNA-dependent; innate immune response; positive regulation of T cell proliferation; regulation of cell growth; positive regulation of T cell activation; inflammatory response; blood coagulation; cell structure disassembly during apoptosis
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. The protein encoded by this gene is one of the PKC family members. It is a calcium-independent and phospholipid-dependent protein kinase. This kinase is important for T-cell activation. It is required for the activation of the transcription factors NF-kappaB and AP-1, and may link the T cell receptor (TCR) signaling complex to the activation of the transcription factors. [provided by RefSeq, Jul 2008]
UniProt Code:	Q04759
NCBI GenInfo Identifier:	334848126
NCBI Gene ID:	5588
NCBI Accession:	NP_001229342.1
UniProt Secondary Accession:	Q04759,Q14DH6, Q3MJF1, Q64FY5, Q9H508, Q9H549,
UniProt Related Accession:	Q04759
Molecular Weight:	81,865 Da
NCBI Full Name:	protein kinase C theta type isoform 2
NCBI Synonym Full Names:	protein kinase C, theta
NCBI Official Symbol:	PRKCQ
NCBI Official Synonym Symbols:	PRKCT; nPKC-theta
NCBI Protein Information:	protein kinase C theta type
UniProt Protein Name:	Protein kinase C theta type
UniProt Synonym Protein Names:	nPKC-theta
UniProt Gene Name:	PRKCQ
UniProt Entry Name:	KPCT_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-PKC theta 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.)