Mouse Midkine (Mdk) ELISA Kit (MOEB0500)- High Sensitivity

Product Name:	Mouse Midkine (Mdk) ELISA Kit
SKU:	MOEB0500
Size:	96T
Target:	Mouse Midkine (Mdk)
Synonyms:	Retanoic acid-responsive protein, Retinoic acid-induced differentiation factor, MK, Mk
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Mouse
Detection Range:	78-5000pg/mL
Sensitivity:	39.6pg/mL

Intra CV:

6.4%

Inter CV:

8.3%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	80-92%	105-115%	110-120%	110-120%
EDTA Plasma(N=5)	81-91%	88-97%	85-97%	82-94%
Heparin Plasma(N=5)	106-117%	104-114%	105-117%	105-115%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	87	81-93
Plasma	89	83-95

Function:

Secreted protein that functions as cytokine and growth factor and mediates its signal through cell-surface proteoglycan and non-proteoglycan receptors (PubMed:16901907). Binds cell-surface proteoglycan receptors via their chondroitin sulfate (CS) groups (PubMed:17230638). Thereby regulates many processes like inflammatory response, cell proliferation, cell adhesion, cell growth, cell survival, tissue regeneration, cell differentiation and cell migration (PubMed:17230638, PubMed:19060126, PubMed:17015789, PubMed:28183532, PubMed:10683378, PubMed:15482347, PubMed:15509530, PubMed:24458438, PubMed:25551381, PubMed:29233575). Participates in inflammatory processes by exerting two different activities. Firstly, mediates neutrophils and macrophages recruitment to the sites of inflammation both by direct action by cooperating namely with ITGB2 via LRP1 and by inducing chemokine expression (PubMed:10683378, PubMed:15509530, PubMed:24458438, PubMed:28183532). This inflammation can be accompanied by epithelial cell survival and smooth muscle cell migration after renal and vessel damage, respectively (PubMed:15509530, PubMed:10683378). Secondly, suppresses the development of tolerogenic dendric cells thereby inhibiting the differentiation of regulatory T cells and also promote T cell expansion through NFAT signaling and Th1 cell differentiation (PubMed:28183532). Promotes tissue regeneration after injury or trauma. After heart damage negatively regulates the recruitment of inflammatory cells and mediates cell survival through activation of anti-apoptotic signaling pathways via MAPKs and AKT pathways through the activation of angiogenesis (PubMed:17015789, PubMed:19060126). Also facilitates liver regeneration as well as bone repair by recruiting macrophage at trauma site and by promoting cartilage development by facilitating chondrocyte differentiation (PubMed:15482347, PubMed:25551381). Plays a role in brain by promoting neural precursor cells survival and growth through interaction with heparan sulfate proteoglycans (PubMed:17230638). Binds PTPRZ1 and promotes neuronal migration and embryonic neurons survival (By similarity). Binds SDC3 or GPC2 and mediates neurite outgrowth and cell adhesion (By similarity). Binds chondroitin sulfate E and heparin leading to inhibition of neuronal cell adhesion induced by binding with GPC2 (PubMed:10978312). Binds CSPG5 and promotes elongation of oligodendroglial precursor-like cells (PubMed:16901907). Also binds ITGA6:ITGB1 complex; this interaction mediates MDK-induced neurite outgrowth (By similarity). Binds LRP1; promotes neuronal survival (By similarity). Binds ITGA4:ITGB1 complex; this interaction mediates MDK-induced osteoblast cells migration through PXN phosphorylation (By similarity). Binds anaplastic lymphoma kinase (ALK) which induces ALK activation and subsequent phosphorylation of the insulin receptor substrate (IRS1), followed by the activation of mitogen-activated protein kinase (MAPK) and PI3-kinase, and the induction of cell proliferation (By similarity). Promotes epithelial to mesenchymal transition through interaction with NOTCH2 (By similarity). During arteriogenesis, plays a role in vascular endothelial cell proliferation by inducing VEGFA expression and release which in turn induces nitric oxide synthase expression. Moreover activates vasodilation through nitric oxide synthase activation (PubMed:29233575). Negatively regulates bone formation in response to mechanical load by inhibiting Wnt/beta-catenin signaling in osteoblasts (PubMed:21185956, PubMed:20200993). In addition plays a role in hippocampal development, working memory, auditory response, early fetal adrenal gland development and the female reproductive system (PubMed:16619002, PubMed:17121547, PubMed:10096022).

Uniprot:	P12025
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant mouse Midkine
Sub Unit:	Homodimer. Interacts with ALK. Interacts with LRP1; promotes neuronal survival. Interacts with LRP2. Interacts with NCAM1 (By similarity). Interacts (via C-terminal) with PTPRZ1 (via chondroitin sulfate chains); this interaction is inhibited by PTN; this interaction promotes neuronal migration (PubMed:10212223). Interacts with NCL; this interaction promotes NCL clustering and lateral movements of this complex into lipid rafts leading to MDK internalization. Interacts with LRP6 and LRP8: this interaction is calcium dependent. Interacts with ITGA4. Interacts with ITGA6. Interacts with ITGB1. Interacts with ITGA4:ITGB1 complex; this interaction mediates MDK-induced osteoblast cells migration through PXN phosphorylation. Interacts with ITGA6:ITGB1 complex; this interaction mediates MDK-induced neurite outgrowth. Interacts with NOTCH2; this interactio mediates a nuclear accumulation of NOTCH2 and therefore activation of NOTCH2 signaling leading to interaction between HES1 and STAT3. Interacts with GPC2 (via heparan sulfate chain); this interaction is inhibited by heparin followed by chondroitin sulfate E; this interaction induces GPC2 clustering through heparan sulfate chain; this interaction induces neuronal cell adhesion and neurite outgrowth (By similarity). Interacts with SDC3; this interaction induces SDC3 clustering; this interaction induces neuronal cell adhesion and neurite outgrowth (PubMed:9089390, PubMed:12084985). Interacts with SDC1 (PubMed:9089390). Interacts with CSPG5; this interaction promotes elongation of oligodendroglial precursor-like cells (PubMed:16901907).
Research Area:	Neurosciences
Subcellular Location:	Secreted
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	MDK: Developmentally regulated, secreted growth factor homologous to pleiotrophin (PTN), which has heparin binding activity. Binds anaplastic lymphoma kinase (ALK) which induces ALK activation and subsequent phosphorylation of the insulin receptor substrate (IRS1), followed by the activation of mitogen-activated protein kinase (MAPK) and PI3-kinase, and the induction of cell proliferation. Involved in neointima formation after arterial injury, possibly by mediating leukocyte recruitment. Also involved in early fetal adrenal gland development. Belongs to the pleiotrophin family.
UniProt Protein Details:	Protein type:Secreted; Secreted, signal peptide Cellular Component: cell projection; cytoplasm; extracellular region Molecular Function:heparin binding; growth factor activity Biological Process: cell migration; behavioral fear response; cerebellar granular layer development; adrenal gland development; short-term memory; multicellular organismal development; positive regulation of transcription, DNA-dependent; hippocampus development; positive regulation of cell division; response to wounding; negative regulation of neuron apoptosis; cell differentiation; defecation; regulation of behavior
NCBI Summary:	This gene encodes a secreted growth factor that belongs to the pleiotrophin/midkine heparin-binding protein family and functions in a variety of biological processes. The encoded cytokine promotes the growth, differentiation, survival and migration of several target cells including leucocytes involved in inflammation. This protein plays a role in the formation of scar tissue and intraperitoneal adhesions, and promotes neurite outgrowth and neuron survival. The protein encoded by this gene is associated with obesity and inhibition of insulin signaling in fat cells. A pseudogene of this gene is present on chromosome 11. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Apr 2014]
UniProt Code:	P12025
NCBI GenInfo Identifier:	127117
NCBI Gene ID:	17242
NCBI Accession:	P12025.2
UniProt Related Accession:	P12025
Molecular Weight:	15,434 Da
NCBI Full Name:	Midkine
NCBI Synonym Full Names:	midkine
NCBI Official Symbol:	Mdk
NCBI Official Synonym Symbols:	MK; Mek
NCBI Protein Information:	midkine; retinoic acid-responsive protein; retinoic acid-induced differentiation factor
UniProt Protein Name:	Midkine
UniProt Synonym Protein Names:	Retinoic acid-induced differentiation factor
Protein Family:	Midkine
UniProt Gene Name:	Mdk
UniProt Entry Name:	MK_MOUSE

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.