Human CSPG4 ELISA Kit (HUEB2202)- High Sensitivity

Product Name:	Human Chondroitin sulfate proteoglycan 4 (CSPG4) ELISA Kit
SKU:	HUEB2202
Size:	96T
Target:	Human Chondroitin sulfate proteoglycan 4 (CSPG4)
Synonyms:	Chondroitin sulfate proteoglycan NG2, Melanoma chondroitin sulfate proteoglycan, Melanoma-associated chondroitin sulfate proteoglycan, MCSP
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.156-10ng/mL
Sensitivity:	0.078ng/mL

Intra CV:

5.8%

Inter CV:

8.4%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	104-114%	93-102%	101-110%	105-115%
EDTA Plasma(N=5)	98-111%	89-98%	107-117%	91-101%
Heparin Plasma(N=5)	96-108%	98-107%	84-94%	98-107%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	92	86-98
Plasma	94	88-100

Function:

Proteoglycan playing a role in cell proliferation and migration which stimulates endothelial cells motility during microvascular morphogenesis. May also inhibit neurite outgrowth and growth cone collapse during axon regeneration. Cell surface receptor for collagen alpha 2(VI) which may confer cells ability to migrate on that substrate. Binds through its extracellular N-terminus growth factors, extracellular matrix proteases modulating their activity. May regulate MPP16-dependent degradation and invasion of type I collagen participating in melanoma cells invasion properties. May modulate the plasminogen system by enhancing plasminogen activation and inhibiting angiostatin. Functions also as a signal transducing protein by binding through its cytoplasmic C-terminus scaffolding and signaling proteins. May promote retraction fiber formation and cell polarization through Rho GTPase activation. May stimulate alpha-4, beta-1 integrin-mediated adhesion and spreading by recruiting and activating a signaling cascade through CDC42, ACK1 and BCAR1. May activate FAK and ERK1/ERK2 signaling cascades.

Uniprot:	Q6UVK1
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Chondroitin sulfate proteoglycan 4
Sub Unit:	Interacts with the first PDZ domain of MPDZ. Interacts with PRKCA. Binds TNC, laminin-1, COL5A1 and COL6A2. Interacts with PLG and angiostatin. Binds FGF2 and PDGFA. Interacts with GRIP1, GRIP2 and GRIA2. Forms a ternary complex with GRIP1 and GRIA2 (By similarity). Interacts with LGALS3 and the integrin composed of ITGB1 and ITGA3. Interacts with ITGA4 through its chondroitin sulfate glycosaminoglycan. Interacts with BCAR1, CDC42 and ACK1. Interacts with MMP16.
Research Area:	Cardiovascular
Subcellular Location:	Cell membrane Single-pass type I membrane protein Extracellular side Apical cell membrane Single-pass type I membrane protein Extracellular side Cell projection Lamellipodium membrane Single-pass type I membrane protein Extracellular side Cell surface Localized at the apical plasma membrane it relocalizes to the lamellipodia of astrocytoma upon phosphorylation by PRKCA. Localizes to the retraction fibers. Localizes to the plasma membrane of oligodendrocytes (By similarity).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	NG2: a transmembrane chondrotin sulfate proteoglycan expressed on several types of immature progenitor cells and human malignant melanoma cells. Plays a role in cell proliferation and motility of endothelial cells during microvascular morphogenesis. May also inhibit neurite outgrowth and growth cone collapse during axon regeneration. Cell surface receptor for collagen alpha 2(VI) which may confer cells ability to migrate on that substrate. May regulate MPP16-dependent degradation and invasion of type I collagen participating to melanoma cells invasion properties. Functions also as a signal transducing protein by binding through its cytoplasmic C-terminus scaffolding and signaling proteins. May promote retraction fiber formation and cell polarization through Rho GTPase activation. May stimulate alpha-4, beta-1 integrin-mediated adhesion and spreading by recruiting and activating a signaling cascade through Cdc42, ACK1 and P130Cas. May activate FAK and ERK signaling cascades. PKC-alpha-mediated NG2 phosphorylation may be a key step for initiating cell polarization and motility.
UniProt Protein Details:	Protein type:Cell adhesion; Membrane protein, integral Chromosomal Location of Human Ortholog: 15q24.2 Cellular Component: lysosomal lumen; cell surface; focal adhesion; integral to plasma membrane; Golgi lumen; apical plasma membrane; extracellular region Molecular Function:signal transducer activity; protein kinase binding Biological Process: chondroitin sulfate biosynthetic process; glycosaminoglycan metabolic process; activation of MAPK activity; tissue remodeling; pathogenesis; glial cell migration; dermatan sulfate biosynthetic process; chondroitin sulfate metabolic process; cell proliferation; positive regulation of peptidyl-tyrosine phosphorylation; carbohydrate metabolic process; chondroitin sulfate catabolic process; angiogenesis; transmembrane receptor protein tyrosine kinase signaling pathway
NCBI Summary:	A human melanoma-associated chondroitin sulfate proteoglycan plays a role in stabilizing cell-substratum interactions during early events of melanoma cell spreading on endothelial basement membranes. CSPG4 represents an integral membrane chondroitin sulfate proteoglycan expressed by human malignant melanoma cells. [provided by RefSeq, Jul 2008]
UniProt Code:	Q6UVK1
NCBI GenInfo Identifier:	296434468
NCBI Gene ID:	1464
NCBI Accession:	Q6UVK1.2
UniProt Related Accession:	Q6UVK1
Molecular Weight:	251kDa
NCBI Full Name:	Chondroitin sulfate proteoglycan 4
NCBI Synonym Full Names:	chondroitin sulfate proteoglycan 4
NCBI Official Symbol:	CSPG4
NCBI Official Synonym Symbols:	NG2; MCSP; MCSPG; MSK16; CSPG4A; HMW-MAA; MEL-CSPG
NCBI Protein Information:	chondroitin sulfate proteoglycan 4
UniProt Protein Name:	Chondroitin sulfate proteoglycan 4
UniProt Synonym Protein Names:	Chondroitin sulfate proteoglycan NG2; Melanoma chondroitin sulfate proteoglycan; Melanoma-associated chondroitin sulfate proteoglycan
Protein Family:	Protein new-glue
UniProt Gene Name:	CSPG4
UniProt Entry Name:	CSPG4_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 MCSP / Melanoma Associated Chondroitin Sulfate Proteoglycan ELISA Kit	Anti-CSPG4 Antibody (CAB3592)[KO Validated]
Human MCSP (Melanoma Associated Chondroitin Sulfate Proteoglycan) CLIA Kit (HUES01257)	CSNK2B Antibody (PACO13416)
	TRERF1 Antibody (PACO20759)
	TRPM7 Antibody (PACO45485)