Mouse Cholecystokinin ELISA Kit- High Sensitivity

Product Name:	Mouse CCK (Cholecystokinin) ELISA Kit
Product Code:	MOFI01216
Size:	96 Assays
Alias:	Cholecystokinin
Detection Method:	Competitive ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse CCK concentrations in serum plasma and other biological fluids.
Sensitivity:	14.063pg/ml
Range:	23.438-1500pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:	Matrices listed below were spiked with certain level of Mouse CCK and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse CCK in samples. Not Available.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse CCK and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Not Available.
Intra Assay:	CV <8%
Inter Assay:	CV <10%

Component	Quantity	Storage
ELISA Microplate(Dismountable)	8-12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	60ul	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)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P09240

UniProt Protein Function:	CCK: This peptide hormone induces gall bladder contraction and the release of pancreatic enzymes in the gut. Its function in the brain is not clear. Binding to CCK-A receptors stimulates amylase release from the pancreas, binding to CCK-B receptors stimulates gastric acid secretion. Belongs to the gastrin/cholecystokinin family. Protein type: Cell development/differentiation; Hormone; Secreted; Secreted, signal peptide Chromosomal Location of Human Ortholog: 9 72.43 cM\|9 F4 Cellular Component: axon; axon hillock; cell soma; dendrite; extracellular region; extracellular space; perikaryon; terminal bouton Molecular Function: hormone activity; neuropeptide hormone activity; protein binding Biological Process: activation of cysteine-type endopeptidase activity involved in apoptotic process; apoptosis; axonogenesis; behavioral fear response; eating behavior; negative regulation of appetite; neuron migration; positive regulation of apoptosis; positive regulation of cell proliferation; positive regulation of glutamate secretion; positive regulation of mitochondrial depolarization; positive regulation of peptidyl-tyrosine phosphorylation; positive regulation of protein oligomerization; protein kinase C-activating G-protein coupled receptor signaling pathway; regulation of sensory perception of pain; release of cytochrome c from mitochondria
UniProt Protein Details:
NCBI Summary:	This gene encodes a member of the gastrin/cholecystokinin family of proteins. The encoded preproprotein is proteolytically processed to generate multiple protein products, including the peptide hormones cholecystokinin-8, -12, -33, and others. The encoded peptides have been shown to regulate gastric acid secretion and food intake. A sulfated form of cholecystokinin-8 may modulate neuronal activity in the brain. Homozygous knockout mice for this gene exhibit impaired insulin secretion, enhanced insulin sensitivity, and resistance to obesity. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Nov 2015]
UniProt Code:	P09240
NCBI GenInfo Identifier:	548962138
NCBI Gene ID:	12424
NCBI Accession:	NP_001271437.1
UniProt Secondary Accession:	P09240,Q53WS9, Q9DCL5
UniProt Related Accession:	P09240
Molecular Weight:
NCBI Full Name:	cholecystokinin isoform 2
NCBI Synonym Full Names:	cholecystokinin
NCBI Official Symbol:	Cck
NCBI Official Synonym Symbols:
NCBI Protein Information:	cholecystokinin
UniProt Protein Name:	Cholecystokinin
UniProt Synonym Protein Names:
Protein Family:	Cholecystokinin
UniProt Gene Name:	Cck
UniProt Entry Name:

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
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.	Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank or Sample per well. The blank well is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody working solution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thorough mixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA platewell, avoid touching plate walls and foaming).
3.	Wash: Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 350µL) using a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC working solution to each well. Cover with a new Plate sealer. Incubate for 30 minutes at 37°C.
5.	Wash: Repeat the aspiration/wash process for five times.
6.	TMB Substrate: Add 90µL of TMB Substrate to each well. Cover with a new Plate sealer. Incubate for about 10-20 minutes at 37°C. Protect from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. When apparent gradient appeared in standard wells, you can terminate the reaction.
7.	Stop: Add 50µL of Stop Solution to each well. Color turn to yellow immediately. The adding order of stop solution should be as the same as the substrate solution.
8.	OD Measurement: Determine the optical density (OD Value) of each well at once, using a microplate reader set to 450 nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters.

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.

Sample Type	Protocol
Serum:	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.
Plasma:	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.
Cell lysates:	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.
Tissue homogenates:	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.
Tissue lysates:	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.
Breast Milk:	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.