CD63 Antibody [P10C2] | Primary Antibodies

Application: Reactivity: Human, Monkey

Lane 1: THP-1, Lane 2: COS-7

Experiment Essentials

WB
Recommended wet transfer conditions: 200 mA, 60 min.

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Human, Monkey

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW
25-60 kDa

Positive Control	THP1 cells; 293T cells; SK-MEL-28 cells; COS-7 cells
Negative Control	Jurkat cells; MOLT-4 cells

Experimental Methods

WB
Experimental Protocol: Sample preparation 1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature. 2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min. 4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant; 5. Remove a small volume of lysate to determine the protein concentration; 6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations 2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.) Transfer membrane 1. Take out the converter, soak the clip and consumables in the pre-cooled converter; 2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer; 3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip"; 4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications Recommended conditions for wet transfer: 200 mA, 60 min. ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.) Block 1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes; 2. Incubate the film in the blocking solution for 1 hour at room temperature; 3. Wash the film with TBST for 3 times, 5 minutes each time. Antibody incubation 1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight; 2. Wash the film with TBST 3 times, 5 minutes each time; 3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour; 4. After incubation, wash the film with TBST 3 times for 5 minutes each time. Antibody staining 1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly; 2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

WB

Experimental Protocol:

Sample preparation

1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature.

2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.

4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;

5. Remove a small volume of lysate to determine the protein concentration;

6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.

Reference Table for Selecting SDS-PAGE Separation Gel Concentrations

2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)

Transfer membrane

1. Take out the converter, soak the clip and consumables in the pre-cooled converter;

2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;

3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";

4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications

Recommended conditions for wet transfer: 200 mA, 60 min.

( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)

Block

1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;

2. Incubate the film in the blocking solution for 1 hour at room temperature;

3. Wash the film with TBST for 3 times, 5 minutes each time.

Antibody incubation

1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;

2. Wash the film with TBST 3 times, 5 minutes each time;

3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;

4. After incubation, wash the film with TBST 3 times for 5 minutes each time.

Antibody staining

1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;

2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.

Datasheet & SDS

Biological Description

Specificity
CD63 Antibody [P10C2] detects endogenous levels of total CD63 protein.

Subcellular Location
Cell membrane, Endosome, Lysosome, Membrane, Secreted

Uniprot ID
P08962

Clone
P10C2

Synonym(s)
CD63 antigen; Granulophysin; Lysosomal-associated membrane protein 3 (LAMP-3); Limp1; Melanoma-associated antigen ME491; OMA81H; Ocular melanoma-associated antigen; Tetraspanin-30 (Tspan-30); CD63; MLA1; TSPAN30

Background
CD63, a (glycosylated) lysosome-associated membrane protein 3 (LAMP3) of the tetraspanin superfamily, is ubiquitously expressed across hematopoietic, endothelial, and melanocytic lineages, where it organizes tetraspanin-enriched microdomains (TEMs) to coordinate integrin signaling, endosomal sorting, and extracellular vesicle (EV) biogenesis. CD63 has four transmembrane domains (TM1–4) forming two tight hairpins, with a small extracellular loop 1 (ECL1, aa 40–70; conserved CCG motif disulfide) and a large ECL2 (aa 113–175; cholesterol-binding palm domain for partner lateral diffusion); cytoplasmic tails (N-term 1–22/C-term 201–237) recruit syntenin-1 and CD47 via dileucine motifs, enabling clathrin/caveolar endocytosis. ESCRT-independent multivesicular endosome (MVE) formation, CD63 scaffolds PMEL luminal domain sorting into intraluminal vesicles (ILVs) of melanosomes to initiate amyloid fibril polymerization (~4-fold fibril increase; CD63 knockdown leads to lysosomal degradation). CD63 also bridges endosomal-autophagy flux, inhibiting mTORC1 (decreasing p-mTOR and retaining LAMTOR1) and regulates EBV LMP1 secretion, balancing signal transduction (CD63 knockout increases LMP1 and mTOR hyperactivation, resulting in large autophagic vacuoles). CD63 is essential for melanosome maturation (PMEL fibrillogenesis), platelet activation (Weibel-Palade body fusion), mast cell degranulation (syntaxin-4 docking), and endothelial permeability (VEGF–integrin-αvβ3 signaling), and is an exosomal marker (~50% EV enrichment). CD63 is upregulated in melanoma, promoting metastasis via TEM–integrin αvβ3/ITGB1 trafficking; CD63 knockout disrupts lysosomal homeostasis, sensitizing cells to lysosomal inhibitors, while paradoxically acting as a tumor suppressor in colon cancer via apoptosis induction.

Background

CD63, a (glycosylated) lysosome-associated membrane protein 3 (LAMP3) of the tetraspanin superfamily, is ubiquitously expressed across hematopoietic, endothelial, and melanocytic lineages, where it organizes tetraspanin-enriched microdomains (TEMs) to coordinate integrin signaling, endosomal sorting, and extracellular vesicle (EV) biogenesis. CD63 has four transmembrane domains (TM1–4) forming two tight hairpins, with a small extracellular loop 1 (ECL1, aa 40–70; conserved CCG motif disulfide) and a large ECL2 (aa 113–175; cholesterol-binding palm domain for partner lateral diffusion); cytoplasmic tails (N-term 1–22/C-term 201–237) recruit syntenin-1 and CD47 via dileucine motifs, enabling clathrin/caveolar endocytosis. ESCRT-independent multivesicular endosome (MVE) formation, CD63 scaffolds PMEL luminal domain sorting into intraluminal vesicles (ILVs) of melanosomes to initiate amyloid fibril polymerization (~4-fold fibril increase; CD63 knockdown leads to lysosomal degradation). CD63 also bridges endosomal-autophagy flux, inhibiting mTORC1 (decreasing p-mTOR and retaining LAMTOR1) and regulates EBV LMP1 secretion, balancing signal transduction (CD63 knockout increases LMP1 and mTOR hyperactivation, resulting in large autophagic vacuoles). CD63 is essential for melanosome maturation (PMEL fibrillogenesis), platelet activation (Weibel-Palade body fusion), mast cell degranulation (syntaxin-4 docking), and endothelial permeability (VEGF–integrin-αvβ3 signaling), and is an exosomal marker (~50% EV enrichment). CD63 is upregulated in melanoma, promoting metastasis via TEM–integrin αvβ3/ITGB1 trafficking; CD63 knockout disrupts lysosomal homeostasis, sensitizing cells to lysosomal inhibitors, while paradoxically acting as a tumor suppressor in colon cancer via apoptosis induction.

References
https://pubmed.ncbi.nlm.nih.gov/29212935/ https://pubmed.ncbi.nlm.nih.gov/21962903/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

Tech Support

Handling Instructions

Tel: +1-832-582-8158 Ext:3

If you have any other enquiries, please leave a message.

Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%