Synuclein-a (Phospho-Ser129) Fluorometric Cell-Based ELISA

Description

Description

Synuclein-a (Phospho-Ser129) Cell-Based ELISA Kit

The Synuclein-a (Phospho-Ser129) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor Synuclein-a phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Synuclein-a in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Synuclein-a phosphorylation.

How does our Synuclein-a (Phospho-Ser129) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of Synuclein-a (Phospho-Ser129) concentration is achieved by an indirect ELISA format. In essence, Synuclein-a (Phospho-Ser129) is captured by Synuclein-a (Phospho-Ser129)-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 Synuclein-a (Phospho-Ser129) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated Synuclein-a can be used to normalize the RFU value for phosphorylated Synuclein-a.

Synuclein-a (Phospho-Ser129) Fluorometric Cell-Based ELISA Kit -Information

Product Name:	Synuclein-a (Phospho-Ser129) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:	FBCAB00034
Description:	The Synuclein-alpha (Phospho-Ser129) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor Synuclein-? (Phospho-Ser129) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Synuclein-? (Phospho-Ser129) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on SNCA 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: 6622, UniProt ID: P37840, OMIM #: 127750/163890/168600/168601/605543, Unigene #: Hs.271771
Format:	Two 96-Well Plates
NCBI Gene Symbol:	SNCA
Sub Type:	Phospho
Target Name:	Phospho-Synuclein-? (Ser129)

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 Synuclein-a (Phospho-Ser129) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total Synuclein-a 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.

Synuclein-a (Phospho-Ser129) - Protein Information

UniProt Protein Function:	SNCA: a member of the synuclein family. Abundantly expressed in the brain. Inhibits phospholipase D2 selectively. May integrate presynaptic signaling and membrane trafficking. Implicated in the pathogenesis of Parkinson's disease. A major component of amyloid plaques in the brains of patients with Alzheimer's disease. Two alternatively spliced isoforms transcripts have been identified.
UniProt Protein Details:	Protein type:Adaptor/scaffold Chromosomal Location of Human Ortholog: 4q21 Cellular Component: Golgi apparatus; nuclear outer membrane; mitochondrion; rough endoplasmic reticulum; lysosome; extracellular region; fibril; terminal button; inclusion body; cell cortex; mitochondrial respiratory chain complex I; cytosol; actin cytoskeleton; synaptic vesicle; platelet alpha granule membrane; growth cone; perinuclear region of cytoplasm; axon; cytoplasm; plasma membrane; ribosome; cell junction; nucleus Molecular Function:protein domain specific binding; identical protein binding; histone binding; zinc ion binding; kinesin binding; ferrous iron binding; microtubule binding; caspase inhibitor activity; beta-tubulin binding; magnesium ion binding; phosphoprotein binding; protein N-terminus binding; oxidoreductase activity; Hsp70 protein binding; calcium ion binding; dynein binding; protein binding; copper ion binding; phospholipase binding; phospholipid binding; tau protein binding; fatty acid binding; alpha-tubulin binding Biological Process: regulation of long-term neuronal synaptic plasticity; negative regulation of serotonin uptake; regulation of acyl-CoA biosynthetic process; adult locomotory behavior; positive regulation of apoptosis; negative regulation of norepinephrine uptake; mitochondrial membrane organization and biogenesis; microglial cell activation; response to lipopolysaccharide; positive regulation of endocytosis; dopamine biosynthetic process; negative regulation of transcription from RNA polymerase II promoter; negative regulation of caspase activity; negative regulation of monooxygenase activity; fatty acid metabolic process; positive regulation of neurotransmitter secretion; regulation of dopamine secretion; negative regulation of dopamine uptake; negative regulation of histone acetylation; calcium ion homeostasis; negative regulation of exocytosis; response to magnesium ion; negative regulation of protein amino acid phosphorylation; behavioral response to cocaine; receptor internalization; phospholipid metabolic process; fibril organization and biogenesis; synapse organization and biogenesis; dopamine uptake; negative regulation of neuron apoptosis; response to iron(II) ion; positive regulation of receptor recycling; aging; caspase activation; response to drug; neutral lipid metabolic process; protein destabilization; regulation of macrophage activation; regulation of glutamate secretion; negative regulation of microtubule polymerization; positive regulation of peptidyl-serine phosphorylation; negative regulation of dopamine metabolic process; organelle ATP synthesis coupled electron transport; regulation of locomotion; synaptic vesicle endocytosis; positive regulation of release of sequestered calcium ion into cytosol; regulation of excitatory postsynaptic membrane potential; negative regulation of transporter activity; negative regulation of apoptosis Disease: Parkinson Disease 4, Autosomal Dominant; Dementia, Lewy Body; Parkinson Disease 1, Autosomal Dominant
NCBI Summary:	Alpha-synuclein is a member of the synuclein family, which also includes beta- and gamma-synuclein. Synucleins are abundantly expressed in the brain and alpha- and beta-synuclein inhibit phospholipase D2 selectively. SNCA may serve to integrate presynaptic signaling and membrane trafficking. Defects in SNCA have been implicated in the pathogenesis of Parkinson disease. SNCA peptides are a major component of amyloid plaques in the brains of patients with Alzheimer's disease. Four alternatively spliced transcripts encoding two different isoforms have been identified for this gene. [provided by RefSeq, Mar 2009]
UniProt Code:	P37840
NCBI GenInfo Identifier:	586067
NCBI Gene ID:	6622
NCBI Accession:	P37840.1
UniProt Secondary Accession:	P37840,Q13701, Q4JHI3, Q6IAU6, A8K2A4,
UniProt Related Accession:	P37840
Molecular Weight:	140
NCBI Full Name:	Alpha-synuclein
NCBI Synonym Full Names:	synuclein, alpha (non A4 component of amyloid precursor)
NCBI Official Symbol:	SNCA
NCBI Official Synonym Symbols:	PD1; NACP; PARK1; PARK4
NCBI Protein Information:	alpha-synuclein; synuclein alpha-140; non A-beta component of AD amyloid
UniProt Protein Name:	Alpha-synuclein
UniProt Synonym Protein Names:	Non-A beta component of AD amyloid; Non-A4 component of amyloid precursor; NACP
Protein Family:	Alpha-synuclein
UniProt Gene Name:	SNCA
UniProt Entry Name:	SYUA_HUMAN