Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection Method:	Colormetric
Detection Range:	0.31-20 ng/mL
Sensitivity:	0.19 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Human CRY1 in samples. No significant cross-reactivity or interference between Human CRY1 and analogues was observed.

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human CRY1. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human CRY1 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 Human CRY1, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human CRY1. The concentration of Human CRY1 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	CRY1: Blue light-dependent regulator of the circadian feedback loop. Inhibits CLOCK\|NPAS2-ARNTL E box-mediated transcription. Acts, in conjunction with CRY2, in maintaining period length and circadian rhythmicity. Has no photolyase activity. Capable of translocating circadian clock core proteins such as PER proteins to the nucleus. May inhibit CLOCK\|NPAS2-ARNTL transcriptional activity through stabilizing the unphosphorylated form of ARNTL. Component of the circadian core oscillator, which includes the CRY proteins, CLOCK or NPAS2, ARNTL or ARNTL2, CSNK1D and/or CSNK1E, TIMELESS, and the PER proteins. Interacts directly with TIMELESS and the PER proteins. Interacts directly with PER1 and PER2 C-terminal domains. Interaction with PER2 inhibits its ubiquitination and vice versa. Binds MAPK. Interacts with FBXL21. Interacts with FBXL3. Expression is regulated by light and circadian rhythms. Peak expression in the suprachiasma nucleus (SCN) and eye at the day/night transition (CT12). Levels decrease with ARNTL-CLOCK inhibition as part of the autoregulatory feedback loop. Belongs to the DNA photolyase class-1 family.
UniProt Protein Details:	Protein type:DNA-binding; Transcription, coactivator/corepressor; Lyase Chromosomal Location of Human Ortholog: 12q23-q24. 1 Cellular Component: mitochondrion; nucleus Molecular Function:ubiquitin binding; histone deacetylase binding; DNA (6-4) photolyase activity; nucleotide binding; transcription factor binding; protein kinase binding; phosphatase binding; protein binding; DNA binding; double-stranded DNA binding; deoxyribodipyrimidine photo-lyase activity; blue light photoreceptor activity; nuclear hormone receptor binding Biological Process: circadian rhythm; response to glucagon stimulus; transcription, DNA-dependent; DNA damage induced protein phosphorylation; negative regulation of G-protein coupled receptor protein signaling pathway; negative regulation of transcription from RNA polymerase II promoter; glucose homeostasis; DNA repair; regulation of circadian rhythm; protein-chromophore linkage; response to insulin stimulus; gluconeogenesis; entrainment of circadian clock by photoperiod; blue light signaling pathway; sequestering of lipid; negative regulation of circadian rhythm; negative regulation of protein ubiquitination; circadian regulation of gene expression; negative regulation of transcription, DNA-dependent
NCBI Summary:	This gene encodes a flavin adenine dinucleotide-binding protein that is a key component of the circadian core oscillator complex, which regulates the circadian clock. This gene is upregulated by CLOCK/ARNTL heterodimers but then represses this upregulation in a feedback loop using PER/CRY heterodimers to interact with CLOCK/ARNTL. Polymorphisms in this gene have been associated with altered sleep patterns. The encoded protein is widely conserved across plants and animals. Loss of the related gene in mouse results in a shortened circadian cycle in complete darkness. [provided by RefSeq, Jan 2014]
UniProt Code:	Q16526
NCBI GenInfo Identifier:	74735764
NCBI Gene ID:	1407
NCBI Accession:	Q16526. 1
UniProt Related Accession:	Q16526
Molecular Weight:	586
NCBI Full Name:	Cryptochrome-1
NCBI Synonym Full Names:	cryptochrome circadian clock 1
NCBI Official Symbol:	CRY1
NCBI Official Synonym Symbols:	PHLL1
NCBI Protein Information:	cryptochrome-1; cryptochrome 1 (photolyase-like)
UniProt Protein Name:	Cryptochrome-1
Protein Family:	Cryptochrome
UniProt Gene Name:	CRY1
UniProt Entry Name:	CRY1_HUMAN

As the OD 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 (ng/mL)	O.D	Average	Corrected
20	2.47 2.52	2.495	2.422
10	1.605 1.613	1.609	1.536
5	0.906 0.884	0.895	0.822
2.5	0.44 0.46	0.45	0.377
1.25	0.249 0.229	0.239	0.166
0.63	0.178 0.168	0.173	0.1
0.31	0.121 0.127	0.124	0.051
0	0.066 0.08	0.073	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human CRY1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human CRY1 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 (ng/mL)	1.07	2.70	8.89	1.02	2.72	8.82
Standard deviation	0.07	0.13	0.49	0.05	0.15	0.28
C V (%)	6.54	4.81	5.51	4.90	5.51	3.17

Recovery

The recovery of Human CRY1 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)	87-100	92
EDTA plasma (n=5)	94-107	101
Cell culture media (n=5)	95-110	101

Linearity

Samples were spiked with high concentrations of Human CRY1 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 (%)	96-111	92-107	97-112
1:2	Average (%)	104	98	103
1:4	Range (%)	94-106	80-92	83-98
1:4	Average (%)	99	86	90
1:8	Range (%)	90-104	84-99	84-94
1:8	Average (%)	96	91	89
1:16	Range (%)	92-109	84-94	90-104
1:16	Average (%)	99	89	95

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 ELISA 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	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
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 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.