Human SNCA (Alpha-synuclein) ELISA Kit - Information
The Assay Genie Human SNCA (Alpha-synuclein) ELISA Kit can assay for Human SNCA in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.How do our Human SNCA (Alpha-synuclein) ELISA Kits Work?
The Assay Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, Assay Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.
At Assay Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound Human SNCA is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.
Human SNCA (Alpha-synuclein) ELISA Kit Data
|Product Code|| |
SNCA, NACP, PARK1, PARK4, PD1, SNCA, Lewy body 4, non A-beta component of AD amyloid, Non-A beta component of AD amyloid, non-A4 component of amyloid, Non-A4 component of amyloid precursor, PARK1, PARK4, synuclein, alpha, non A4 component of amyloid precursor, Synuclein-alpha
|Detection method|| |
Sandwich ELISA, Double Antibody
This immunoassay kit allows for the in vitro quantitative determination of Human SNCA concentrations in serum plasma and other biological fluids.
4'C for 6 months
Matrices listed below were spiked with certain level of Human SNCA and the recovery rates were calculated by comparing the measured value to the expected amount of Human SNCA in samples.
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human SNCA and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
For Research Use Only
Human SNCA (Alpha-synuclein) ELISA Kit Protocol
The below protocol is a sample protocol for Human SNCA (Alpha-synuclein) ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISA Kits allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human SNCA Antibody present in their sample.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
|1.||Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!|
|2.||Aliquot 0.1ml standard solutions into the standard wells.|
|3.||Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.|
|4.||Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.|
|5.||Seal the plate with a cover and incubate at 37 °C for 90 min.|
|6.||Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.|
|7.||Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.|
|8.||Seal the plate with a cover and incubate at 37°C for 60 min.|
|9.||Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.|
|10.||Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.|
|11.||Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.|
|12.||Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.|
|13.||Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.|
|14.||Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.|
Human SNCA (Alpha-synuclein) ELISA Kit components
|ELISA Microplate (Dismountable)||8×12 strips||4°C for 6 months|
|Sample/Standard Dilution Buffer||20ml||4°C|
|Biotin-labeled Antibody(Concentrated)||120ul||4°C (Protect from light)|
|Antibody Dilution Buffer||10ml||4°C|
|HRP-Streptavidin Conjugate(SABC)||120ul||4°C (Protect from light)|
|SABC Dilution Buffer||10ml||4°C|
|TMB Substrate||10ml||4°C (Protect from light)|
Other materials and equipment required:The Assay Genie Human SNCA (Alpha-synuclein) ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid
Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant
Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.
Human SNCA 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:|
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]|
|NCBI GenInfo Identifier:||586067|
|NCBI Gene ID:||6622|
|UniProt Secondary Accession:||P37840,Q13701, Q4JHI3, Q6IAU6, A8K2A4,|
|UniProt Related Accession:||P37840|
|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|
|UniProt Gene Name:||SNCA|
|UniProt Entry Name:||SYUA_HUMAN|