인간 Ryanodine receptor 2 (RYR2) ELISA Kit

Product Name:	Human Ryanodine receptor 2 (RYR2) ELISA Kit
Product Code:	HUEB1458
Alias:	Ryanodine receptor 2, RYR-2, RyR2, hRYR-2, Cardiac muscle ryanodine receptor-calcium release channel, Cardiac muscle-type ryanodine receptor, RYR2, Type 2 ryanodine receptor
Uniprot:	Q92736
Reactivity:	Human
Range:	0.625-40 ng/mL
Detection Method:	Sandwich
Size:	96 Assay
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	RYR2: ryanodine receptor calcium release channel. Regulates communication between transverse-tubules and sarcoplasmic reticulum. Contraction of cardiac muscle is triggered by release of calcium ions from SR following depolarization of T-tubules. Phosphorylation by PKA dissociates FKBP12.6 from RYR2. Two splice isoforms have been described.
UniProt Protein Details:	Protein type:Transporter, ion channel; Membrane protein, integral; Membrane protein, multi-pass; Channel, ligand-gated; Transporter Chromosomal Location of Human Ortholog: 1q43 Cellular Component: sarcoplasmic reticulum membrane; smooth endoplasmic reticulum; protein complex; membrane; sarcoplasmic reticulum; plasma membrane; Z disc Molecular Function:calcium-induced calcium release activity; calmodulin binding; identical protein binding; protein binding; enzyme binding; protein self-association; calcium-release channel activity; calcium channel activity; calcium ion binding; intracellular ligand-gated calcium channel activity; ryanodine-sensitive calcium-release channel activity Biological Process: positive regulation of sequestering of calcium ion; cytosolic calcium ion homeostasis; regulation of heart rate; calcium-mediated signaling; positive regulation of heart rate; response to redox state; Wnt receptor signaling pathway through beta-catenin; release of sequestered calcium ion by sarcoplasmic reticulum into cytosol; response to caffeine; cellular calcium ion homeostasis; BMP signaling pathway; calcium ion transport; release of sequestered calcium ion into cytosol; response to hypoxia; detection of calcium ion; transmembrane transport; cardiac muscle contraction Disease: Ventricular Tachycardia, Catecholaminergic Polymorphic, 1, With Or Without Atrial Dysfunction And/or Dilated Cardiomyopathy; Arrhythmogenic Right Ventricular Dysplasia, Familial, 2
NCBI Summary:	This gene encodes a ryanodine receptor found in cardiac muscle sarcoplasmic reticulum. The encoded protein is one of the components of a calcium channel, composed of a tetramer of the ryanodine receptor proteins and a tetramer of FK506 binding protein 1B proteins, that supplies calcium to cardiac muscle. Mutations in this gene are associated with stress-induced polymorphic ventricular tachycardia and arrhythmogenic right ventricular dysplasia. [provided by RefSeq, Jul 2008]
UniProt Code:	Q92736
NCBI GenInfo Identifier:	308153558
NCBI Gene ID:	6262
NCBI Accession:	Q92736.3
UniProt Secondary Accession:	Q92736,Q15411, Q546N8, Q5T3P2,
UniProt Related Accession:	Q92736
Molecular Weight:	565kDa
NCBI Full Name:	Ryanodine receptor 2
NCBI Synonym Full Names:	ryanodine receptor 2 (cardiac)
NCBI Official Symbol:	RYR2
NCBI Official Synonym Symbols:	RyR; ARVC2; ARVD2; RYR-2; VTSIP
NCBI Protein Information:	ryanodine receptor 2; type 2 ryanodine receptor; islet-type ryanodine receptor; kidney-type ryanodine receptor; cardiac-type ryanodine receptor; cardiac muscle ryanodine receptor-calcium release channel
UniProt Protein Name:	Ryanodine receptor 2
UniProt Synonym Protein Names:	Cardiac muscle ryanodine receptor; Cardiac muscle ryanodine receptor-calcium release channel; Type 2 ryanodine receptor
Protein Family:	Ryanodine receptor
UniProt Gene Name:	RYR2
UniProt Entry Name:	RYR2_HUMAN

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

ELISA

Human Ryanodine receptor 2 / RYR2 ELISA Kit