Human RIPK3 ELISA Kit (HUEB1123)- High Sensitivity

Product Name:	Human Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) ELISA Kit
SKU:	HUEB1123
Size:	96T
Target:	Human Receptor-interacting serine/threonine-protein kinase 3 (RIPK3)
Synonyms:	RIP-like protein kinase 3, Receptor-interacting protein 3, RIP-3, RIP3
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.156-10ng/mL
Sensitivity:	0.086ng/mL

Intra CV:

5.1%

Inter CV:

7.4%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	87-96%	97-106%	110-119%	111-123%
EDTA Plasma(N=5)	92-104%	110-119%	102-110%	111-120%
Heparin Plasma(N=5)	106-115%	106-117%	90-100%	104-114%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	82	80-88
Plasma	84	80-90

Function:

Essential for necroptosis, a programmed cell death process in response to death-inducing TNF-alpha family members. Upon induction of necrosis, RIPK3 interacts with, and phosphorylates RIPK1 and MLKL to form a necrosis-inducing complex. RIPK3 binds to and enhances the activity of three metabolic enzymes: GLUL, GLUD1, and PYGL. These metabolic enzymes may eventually stimulate the tricarboxylic acid cycle and oxidative phosphorylation, which could result in enhanced ROS production.

Uniprot:	Q9Y572
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Receptor-interacting serine/threonine-protein kinase 3
Sub Unit:	Interacts (via RIP homotypic interaction motif) with RIPK1 (via RIP homotypic interaction motif); this interaction induces RIPK1 phosphorylation and formation of a RIPK1-RIPK3 necrosis-inducing complex. Upon TNF-induced necrosis, the RIPK1-RIPK3 dimer further interacts with PGAM5 and MLKL; the formation of this complex leads to PGAM5 phosphorylation and increase in PGAM5 phosphatase activity. Interacts with MLKL; the interaction is direct. Binds TRAF2 and is recruited to the TNFR-1 signaling complex. Interacts with PYGL, GLUL and GLUD1; these interactions result in activation of these metabolic enzymes. Interacts with BIRC2/c-IAP1, BIRC3/c-IAP2 and XIAP/BIRC4. Interacts with ARHGEF2.
Research Area:	Immunology
Subcellular Location:	Cytoplasm Cytosol Cell membrane Mitochondrion
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	RIPK3: Promotes apoptosis. Belongs to the protein kinase superfamily. TKL Ser/Thr protein kinase family. Binds TRAF2 and RIPK1 and is recruited to the TNFR-1 signaling complex. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:EC 2.7.11.1; Protein kinase, Ser/Thr (non-receptor); Protein kinase, TKL; Kinase, protein; TKL group; RIPK family Chromosomal Location of Human Ortholog: 14q11.2 Cellular Component: mitochondrion; plasma membrane; cytosol Molecular Function:NF-kappaB-inducing kinase activity; protein serine/threonine kinase activity; identical protein binding; protein binding; transcription coactivator activity; protein complex binding; ATP binding; protein kinase activity Biological Process: spleen development; I-kappaB kinase/NF-kappaB cascade; regulation of activated T cell proliferation; thymus development; protein heterooligomerization; protein amino acid autophosphorylation; MyD88-independent toll-like receptor signaling pathway; T cell differentiation in the thymus; protein modification process; toll-like receptor 3 signaling pathway; signal transduction; lymph node development; activation of NF-kappaB transcription factor; regulation of T cell mediated cytotoxicity; regulation of adaptive immune response; regulation of interferon-gamma production; toll-like receptor signaling pathway; positive regulation of interferon type I production; activation of protein kinase activity; innate immune response; T cell homeostasis; positive regulation of ligase activity; toll-like receptor 4 signaling pathway; protein homooligomerization; positive regulation of oxidoreductase activity
UniProt Code:	Q9Y572
NCBI GenInfo Identifier:	40254844
NCBI Gene ID:	11035
NCBI Accession:	NP_006862
UniProt Secondary Accession:	Q9Y572,Q5J795, Q5J796, Q6P5Y1, B4DJL9, C4AM87,
UniProt Related Accession:	Q9Y572
Molecular Weight:	25,326 Da
NCBI Full Name:	receptor-interacting serine/threonine-protein kinase 3
NCBI Synonym Full Names:	receptor-interacting serine-threonine kinase 3
NCBI Official Symbol:	RIPK3
NCBI Official Synonym Symbols:	RIP3
NCBI Protein Information:	receptor-interacting serine/threonine-protein kinase 3; RIP-3; RIP-like protein kinase 3; receptor interacting protein 3; receptor-interacting protein 3
UniProt Protein Name:	Receptor-interacting serine/threonine-protein kinase 3
UniProt Synonym Protein Names:	RIP-like protein kinase 3; Receptor-interacting protein 3; RIP-3
Protein Family:	Putative zinc metalloprotease
UniProt Gene Name:	RIPK3
UniProt Entry Name:	RIPK3_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	Antibodies
Human RIPK3 (Receptor-interacting serine/threonine-protein kinase 3) ELISA Kit (HUFI04910)	Anti-RIPK3 Antibody (CAB5431)
	RIPK2 Antibody (PACO11850)
	FARP1 Antibody, Biotin conjugated (PACO42841)
	RIPK3 Antibody (PACO42842)
	RIPK3 Antibody, HRP conjugated (PACO42843)