Human CD63 antigen (CD63) ELISA Kit- High Sensitivity

Product Name:	Human CD63 antigen (CD63) ELISA Kit
SKU:	HUEB2641
Size:	96T
Target:	Human CD63 antigen (CD63)
Synonyms:	Granulophysin, Lysosomal-associated membrane protein 3, Melanoma-associated antigen ME491, OMA81H, Ocular melanoma-associated antigen, Tetraspanin-30, LAMP-3, Tspan-30, CD63, MLA1, TSPAN30
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	31.2-2000pg/ml
Sensitivity:	19.5pg/mL

Intra CV:

5.8%

Inter CV:

11.0%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	92-102%	100-109%	97-108%	119-119%
EDTA Plasma(N=5)	83-93%	108-120%	100-110%	86-96%
Heparin Plasma(N=5)	102-112%	106-115%	85-95%	92-104%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	88	82-94
Plasma	90	84-96

Function:

Functions as cell surface receptor for TIMP1 and plays a role in the activation of cellular signaling cascades. Plays a role in the activation of ITGB1 and integrin signaling, leading to the activation of AKT, FAK/PTK2 and MAP kinases. Promotes cell survival, reorganization of the actin cytoskeleton, cell adhesion, spreading and migration, via its role in the activation of AKT and FAK/PTK2. Plays a role in VEGFA signaling via its role in regulating the internalization of KDR/VEGFR2. Plays a role in intracellular vesicular transport processes, and is required for normal trafficking of the PMEL luminal domain that is essential for the development and maturation of melanocytes. Plays a role in the adhesion of leukocytes onto endothelial cells via its role in the regulation of SELP trafficking. May play a role in mast cell degranulation in response to Ms4a2/FceRI stimulation, but not in mast cell degranulation in response to other stimuli.

Uniprot:	P08962
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human CD63 antigen
Sub Unit:	Interacts with TIMP1 and ITGB1 and recruits TIMP1 to ITGB1 (PubMed:16917503, PubMed:24635319). Interacts with CD9. Identified in a complex with CD9 and ITGB3 (PubMed:19640571). Interacts with PMEL (PubMed:21962903). Interacts with KDR/VEGFR2; identified in a complex with ITGB1 and KDR/VEGFR2 and is required to recruit KDR to ITGB1 complexes (PubMed:23632027). Interacts with SYT7.
Research Area:	Cancer
Subcellular Location:	Cell membrane Multi-pass membrane protein Lysosome membrane Multi-pass membrane protein Late endosome membrane Multi-pass membrane protein Endosome Multivesicular body Melanosome Secreted Exosome Cell surface Also found in Weibel-Palade bodies of endothelial cells (PubMed:10793155). Located in platelet dense granules (PubMed:7682577). Detected in a subset of pre-melanosomes. Detected on intralumenal vesicles (ILVs) within multivesicular bodies (PubMed:21962903).
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	Function: This antigen is associated with early stages of melanoma tumor progression. May play a role in growth regulation.
UniProt Protein Details:	Subcellular location: Cell membrane; Multi-pass membrane protein. Lysosome membrane; Multi-pass membrane protein. Late endosome membrane; Multi-pass membrane protein. Note: Also found in Weibel-Palade bodies of endothelial cells. Located in platelet dense granules. Ref.3 Ref.13 Ref.15 Ref.17 Ref.18 Tissue specificity: Dysplastic nevi, radial growth phase primary melanomas, hematopoietic cells, tissue macrophages. Miscellaneous: Lack of expression of CD63 in platelets has been observed in a patient with Hermansky-Pudlak syndrome (HPS). Hermansky-Pudlak syndrome (HPS) is a genetically heterogeneous, rare, autosomal recessive disorder characterized by oculocutaneous albinism, bleeding due to platelet storage pool deficiency, and lysosomal storage defects. This syndrome results from defects of diverse cytoplasmic organelles including melanosomes, platelet dense granules and lysosomes. Ceroid storage in the lungs is associated with pulmonary fibrosis, a common cause of premature death in individuals with HPS. Sequence similarities: Belongs to the tetraspanin (TM4SF) family.
NCBI Summary:	The protein encoded by this gene is a member of the transmembrane 4 superfamily, also known as the tetraspanin family. Most of these members are cell-surface proteins that are characterized by the presence of four hydrophobic domains. The proteins mediate signal transduction events that play a role in the regulation of cell development, activation, growth and motility. The encoded protein is a cell surface glycoprotein that is known to complex with integrins. It may function as a blood platelet activation marker. Deficiency of this protein is associated with Hermansky-Pudlak syndrome. Also this gene has been associated with tumor progression. Alternative splicing results in multiple transcript variants encoding different protein isoforms. [provided by RefSeq, Apr 2012]
UniProt Code:	P08962
NCBI GenInfo Identifier:	383872547
NCBI Gene ID:	967
NCBI Accession:	NP_001244330.1
UniProt Secondary Accession:	P08962,Q5TZP3, Q8N6Z9, Q9UCG6, F8VZE2,
UniProt Related Accession:	P08962
Molecular Weight:
NCBI Full Name:	CD63 antigen isoform D
NCBI Synonym Full Names:	CD63 molecule
NCBI Official Symbol:	CD63
NCBI Official Synonym Symbols:	MLA1; ME491; LAMP-3; OMA81H; TSPAN30
NCBI Protein Information:	CD63 antigen; tspan-30; granulophysin; tetraspanin-30; melanoma-associated antigen MLA1; CD63 antigen (melanoma 1 antigen); melanoma-associated antigen ME491; ocular melanoma-associated antigen; lysosomal-associated membrane protein 3; lysosome-associated membrane glycoprotein 3
UniProt Protein Name:	CD63 antigen
UniProt Synonym Protein Names:	Granulophysin; Lysosomal-associated membrane protein 3; LAMP-3; Melanoma-associated antigen ME491; OMA81H; Ocular melanoma-associated antigen; Tetraspanin-30
Protein Family:	CD63 antigen
UniProt Gene Name:	CD63
UniProt Entry Name:	CD63_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 CD63 / TSPAN30 ELISA Kit	Anti-CD63 Antibody (CAB19023)
Human TSPAN30/CD63 (Tetraspanin 30) CLIA Kit (HUES01268)	Anti-CD63 Antibody (CAB5271)
	CD63 Antibody (PACO02193)
	EIF5B Antibody (PACO05800)
	OLR1 Antibody (PACO18688)