PLCG1 (Phospho-Tyr783) Fluorometric Cell-Based ELISA Kit

SKU:

FBCAB00014

Product Type:

ELISA Kit

ELISA Type:

Cell Based Phospho Specific

Reactivity:

Human

Mouse

Rat

Detection Method:

Fluorometric

Description

Description

PLCG1 (Phospho-Tyr783) Cell-Based ELISA Kit

The PLCG1 (Phospho-Tyr783) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor PLCG1 phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated PLCG1 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on PLCG1 phosphorylation.

How does our PLCG1 (Phospho-Tyr783) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of PLCG1 (Phospho-Tyr783) concentration is achieved by an indirect ELISA format. In essence, PLCG1 (Phospho-Tyr783) is captured by PLCG1 (Phospho-Tyr783)-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 PLCG1 (Phospho-Tyr783) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated PLCG1 can be used to normalize the RFU value for phosphorylated PLCG1.

PLCG1 (Phospho-Tyr783) Fluorometric Cell-Based ELISA Kit -Information

Product Name:	PLCG1 (Phospho-Tyr783) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:	FBCAB00014
Description:	The PLCG1 (Phospho-Tyr783) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor PLCG1 (Phospho-Tyr783) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated PLCG1 (Phospho-Tyr783) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on PLCG1 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: 5335, UniProt ID: P19174, OMIM #: 172420, Unigene #: Hs.268177
Format:	Two 96-Well Plates
NCBI Gene Symbol:	PLCG1
Sub Type:	Phospho
Target Name:	Phospho-PLCG1 (Tyr783)

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 PLCG1 (Phospho-Tyr783) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total PLCG1 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.

PLCG1 (Phospho-Tyr783) - Protein Information

UniProt Protein Function:	PLCG1: a calcium dependent phosphatidylinositol-specific phospholipase C. The activated enzyme produces the second messenger molecules diacylglycerol and inositol 1,4,5-trisphosphate. Phosphorylated and activated by tyrosine kinases in response to signaling through a variety of growth factor receptors and immune system receptors.
UniProt Protein Details:	Protein type:Motility/polarity/chemotaxis; Phospholipase; Carbohydrate Metabolism - inositol phosphate; EC 3.1.4.11 Chromosomal Location of Human Ortholog: 20q12-q13.1 Cellular Component: cell projection; cytoplasm; cytosol; intercellular junction; lamellipodium; plasma membrane; ruffle; signalosome Molecular Function:calcium ion binding; glutamate receptor binding; neurotrophin TRKA receptor binding; phosphoinositide phospholipase C activity; phospholipase C activity; protein binding; protein kinase binding; receptor signaling protein activity; receptor tyrosine kinase binding Biological Process: activation of MAPKK activity; axon guidance; blood coagulation; calcium-mediated signaling; cell migration; cytokine and chemokine mediated signaling pathway; epidermal growth factor receptor signaling pathway; fibroblast growth factor receptor signaling pathway; in utero embryonic development; innate immune response; inositol phosphate metabolic process; leukocyte migration; nerve growth factor receptor signaling pathway; phospholipase C activation; phospholipid catabolic process; positive regulation of angiogenesis; positive regulation of blood vessel endothelial cell migration; positive regulation of release of sequestered calcium ion into cytosol; signal transduction; T cell receptor signaling pathway; vascular endothelial growth factor receptor signaling pathway; viral reproduction
NCBI Summary:	The protein encoded by this gene catalyzes the formation of inositol 1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. This reaction uses calcium as a cofactor and plays an important role in the intracellular transduction of receptor-mediated tyrosine kinase activators. For example, when activated by SRC, the encoded protein causes the Ras guanine nucleotide exchange factor RasGRP1 to translocate to the Golgi, where it activates Ras. Also, this protein has been shown to be a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Jul 2008]
UniProt Code:	P19174
NCBI GenInfo Identifier:	130225
NCBI Gene ID:	5335
NCBI Accession:	P19174.1
UniProt Secondary Accession:	P19174,Q2V575, B7ZLY7, B9EGH4, E1P5W4,
UniProt Related Accession:	P19174
Molecular Weight:	148,660 Da
NCBI Full Name:	1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1
NCBI Synonym Full Names:	phospholipase C gamma 1
NCBI Official Symbol:	PLCG1
NCBI Official Synonym Symbols:	PLC1; NCKAP3; PLC-II; PLC148; PLCgamma1
NCBI Protein Information:	1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1
UniProt Protein Name:	1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase gamma-1
UniProt Synonym Protein Names:	PLC-148; Phosphoinositide phospholipase C-gamma-1; Phospholipase C-II; PLC-II; Phospholipase C-gamma-1; PLC-gamma-1
Protein Family:	1-phosphatidylinositol 4,5-bisphosphate phosphodiesterase
UniProt Gene Name:	PLCG1
UniProt Entry Name:	PLCG1_HUMAN