PKC theta (Phospho-Thr538) Fluorometric Cell-Based ELISA

PKC theta (Phospho-Thr538) Fluorometric Cell-Based ELISA Kit

SKU:

FBCAB00044

Product Type:

ELISA Kit

ELISA Type:

Cell Based Phospho Specific

Reactivity:

Human

Mouse

Rat

Detection Method:

Fluorometric

Description

Description

PKC theta (Phospho-Thr538) Cell-Based ELISA Kit

The PKC theta (Phospho-Thr538) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor PKC theta phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated 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 phosphorylation.

How does our PKC theta (Phospho-Thr538) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of PKC theta (Phospho-Thr538) concentration is achieved by an indirect ELISA format. In essence, PKC theta (Phospho-Thr538) is captured by PKC theta (Phospho-Thr538)-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. 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 RFU values.
2.	An antibody against the nonphosphorylated counterpart of PKC theta (Phospho-Thr538) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated PKC theta can be used to normalize the RFU value for phosphorylated PKC theta.

PKC theta (Phospho-Thr538) Fluorometric Cell-Based ELISA Kit -Information

Product Name:	PKC theta (Phospho-Thr538) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:	FBCAB00044
Description:	The PKC theta (Phospho-Thr538) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor PKC ? (Phospho-Thr538) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated PKC ? (Phospho-Thr538) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on PRKCQ phosphorylation.
Dynamic Range:	> 5000 Cells
Detection Method:	Fluorometric
Storage/Stability:	4°C/6 Months
Reactivity:	Human, Mouse, Rat
Assay Type:	Cell-Based ELISA
Database Links:	Gene ID: 5588, UniProt ID: Q04759, OMIM #: 600448, Unigene #: Hs.498570
Format:	Two 96-Well Plates
NCBI Gene Symbol:	PRKCQ
Sub Type:	Phospho
Target Name:	Phospho-PKC ? (Thr538)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components	Quantity
96-Well Black Cell CultureClear-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	110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody	6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody	6 ml
Adhesive Plate Seals	2 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:

Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
Micropipettes capable of measuring volumes from 1 µl to 1 ml
Deionized or sterile water (ddH2O)
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)

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 desired cell concentration in culture medium into each well of the 96-well plates. 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 3 minutes. 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 3 minutes each time.
9.	Dispense 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 3 minutes each time.
11.	Add 50 µl of Primary Antibody Mixture P to corresponding wells for PKC theta (Phospho-Thr538) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total PKC theta detection. Cover the plate 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 3 minutes each time.
13.	Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.	Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.	Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.	Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

PKC theta (Phospho-Thr538) - Protein Information

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