Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Rat
Detection method:	Chemiluminescence
Detection range:	31.25-2000 pg/mL
Sensitivity:	18.75 pg/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Rat SNAP-25 in samples. No significant cross-reactivity or interference between Rat SNAP-25 and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Rat SNAP-25. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat SNAP-25 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Rat SNAP-25, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Rat SNAP-25. The concentration of Rat SNAP-25 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	SNAP-25: a presynaptic plasma membrane protein involved in the regulation of neurotransmitter release. May play an important role in the synaptic function of specific neuronal systems. Part of the SNARE core complex containing SNAP25, VAMP2 and syntaxin 1A. Associates with proteins involved in vesicle docking and membrane fusion. Two alternatively spliced isoforms have been described.
UniProt Protein Details:	Protein type:Vesicle Cellular Component: voltage-gated potassium channel complex; neuron projection; cell; terminal button; intracellular organelle; trans-Golgi network; cytosol; actin cytoskeleton; lipid raft; SNARE complex; growth cone; cell soma; membrane; lamellipodium; axon; perinuclear region of cytoplasm; cytoplasm; plasma membrane; synapse; cell junction; vesicle; myelin sheath; endosome; filopodium Molecular Function:SNAP receptor activity; protein domain specific binding; voltage-gated potassium channel activity; protein binding; SNARE binding; myosin binding; syntaxin-1 binding; protein N-terminus binding; calcium-dependent protein binding Biological Process: synaptic vesicle fusion to presynaptic membrane; growth hormone secretion; exocytosis; axonogenesis; long-term memory; regulation of synaptogenesis; neurotransmitter secretion; calcium ion-dependent exocytosis of neurotransmitter; positive regulation of hormone secretion; positive regulation of insulin secretion; endosome transport; sleep; synaptic vesicle priming
NCBI Summary:	Synaptic vesicle membrane docking and fusion is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) located on the vesicle membrane (v-SNAREs) and the target membrane (t-SNAREs). The assembled v-SNARE/t-SNARE complex consists of a bundle of four helices, one of which is supplied by v-SNARE and the other three by t-SNARE. For t-SNAREs on the plasma membrane, the protein syntaxin supplies one helix and the protein encoded by this gene contributes the other two. Therefore, this gene product is a presynaptic plasma membrane protein. It is essential for regulated exocytosis in neuronal cells. Alternative transcript variants encoding two different protein isoforms have been described for this gene. [provided by RefSeq, Jul 2012]
UniProt Code:	P60881
NCBI GenInfo Identifier:	396080316
NCBI Gene ID:	25012
NCBI Accession:	NP_001257505. 1
UniProt Secondary Accession:	P60881,P13795, P36974, P70557, P70558, Q8IXK3, Q96FM2 Q9BR45,
UniProt Related Accession:	P60881
Molecular Weight:
NCBI Full Name:	synaptosomal-associated protein 25 isoform a
NCBI Synonym Full Names:	synaptosomal-associated protein 25
NCBI Official Symbol:	Snap25
NCBI Official Synonym Symbols:	SNAP-25B; SNAP-25a
NCBI Protein Information:	synaptosomal-associated protein 25; SUP; SNAP-25; super protein; synaptosomal-associated 25 kDa protein; synaptosomal-associated protein, 25 kDa
UniProt Protein Name:	Synaptosomal-associated protein 25
UniProt Synonym Protein Names:	Super protein; SUP; Synaptosomal-associated 25 kDa protein
Protein Family:	Synaptosomal-associated protein
UniProt Gene Name:	Snap25
UniProt Entry Name:	SNP25_RAT

As the RLU values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

Concentration (pg/mL)	RLU	Average	Corrected
2000	55418 56024	55721	55693
1000	21869 26079	23974	23946
500	11822 10242	11032	11004
250	4824 5764	5294	5266
125	2746 2470	2608	2580
62.5	1371 1251	1311	1283
31.25	655 693	674	646
0	27 29	28	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Rat SNAP-25 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Rat SNAP-25 were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (pg/mL)	110.86	210.84	974.52	100.78	193.61	949.85
Standard deviation	14.06	21.95	98.91	8.91	18.41	59.75
C V (%)	12.68	10.41	10.15	8.84	9.51	6.29

Recovery

The recovery of Rat SNAP-25 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type	Range (%)	Average Recovery (%)
Serum (n=5)	87-98	92
EDTA plasma (n=5)	98-114	106
Cell culture media (n=5)	100-115	107

Linearity

Samples were spiked with high concentrations of Rat SNAP-25 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

		Serum (n=5)	EDTA plasma (n=5)	Cell culture media (n=5)
1:2	Range (%)	86-98	94-108	100-116
1:2	Average (%)	92	101	106
1:4	Range (%)	98-113	88-101	87-99
1:4	Average (%)	106	93	94
1:8	Range (%)	97-112	94-106	97-112
1:8	Average (%)	104	100	103
1:16	Range (%)	92-106	85-98	87-101
1:16	Average (%)	97	92	94

An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item	Specifications	Storage
Micro CLIA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.