CD63 Rabbit mAb

Catalog No.: F0983

Immunohistochemical analysis of formalin fixed paraffin embedded human Colorectal cancer tissue with F0983 at 1/100 dilution.

1/2

サイズ（液体）	価格(税別)	在庫状況
50uL	JPY 23500	国内在庫なし(納期7~10日)
100uL	JPY 35500	国内在庫なし(納期7~10日)
100uL*2	JPY 53500	お問い合わせ

代表番号: 045-509-1970\|電子メール：sales@selleck.co.jp よく尋ねられる質問

文献中Selleckの製品使用例(1)

Stem Cell Res Ther,202314(1):176

カスタマーフィードバック(2)

キーポイント

タンパク質の局在：細胞膜，リソソーム，エンドソーム，細胞外環境。
WB
RIPA/NP-40 Lysis Buffer バッファーでのライセート調製を推奨します。
転写条件（ウェット）: 200 mA, 60 min。

使用情報

Dilution
WB1:1000 IHC1:200

Application
WB, IHC

Source
Rabbit

Reactivity
Human

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
26-60 kDa

ポジティブコントロール	HAP1 (Brefeldin A treated); A375; HL-60; Human tonsil; Human platelet; Human melanoma
ネガティブコントロール	Human skeletal muscle

サンプル処理データの例

サンプル	処理状況
HAP1	Brefeldin A-treated (10 µ/mL, 24 h)
HepG2	Brefeldin A-treated (10 µ/mL, 24 h)
クリックして、さらに多くのサンプルデータを表示

^*異なるヒト由来細胞や組織における発現量の予測については、以下をご参照ください: http://www.proteinatlas.org

サンプル	処理状況

プロトコール

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. Electrophoretic separation 1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. 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.

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.

Electrophoretic separation

1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 10%. 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.

IF
Experimental Protocol： Specimen Preparation 1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS. NOTE: Paraformaldehyde is toxic, use only in a fume hood. 2. Fix cells for 15 min at room temperature. 3. Aspirate fixative, rinse three times in 1X PBS for 5 min each. 4. Proceed with Immunostaining. Immunostaining 1. Add theblocking buffer and incubate for 60 min at RT. 2. Prepare primary antibody diluent in antibody dilution buffer as recommended . 3. Aspirate blocking solution, apply diluted primary antibody. 4. Incubate overnight at 4°C. 5. Rinse three times in 1X PBS for 5 min each. 6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark. 7. Rinse three times in 1X PBS for 5 min each. 8. Mount slides usingmounting medium with DAPI and cover with coverslips. 9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

Experimental Protocol：

Specimen Preparation

1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS.

NOTE: Paraformaldehyde is toxic, use only in a fume hood.

2. Fix cells for 15 min at room temperature.

3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.

4. Proceed with Immunostaining.

Immunostaining

1. Add theblocking buffer and incubate for 60 min at RT.

2. Prepare primary antibody diluent in antibody dilution buffer as recommended .

3. Aspirate blocking solution, apply diluted primary antibody.

4. Incubate overnight at 4°C.

5. Rinse three times in 1X PBS for 5 min each.

6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.

7. Rinse three times in 1X PBS for 5 min each.

8. Mount slides usingmounting medium with DAPI and cover with coverslips.

9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.

Datasheet & SDS

生物学的記述

Specificity
CD63 Rabbit mAb detects endogenous levels of total CD63 protein.

Uniprot ID
P08962

Clone
N10M7

Background
CD63 is a member of the tetraspanin family, distinguished by its four transmembrane domains, a short extracellular domain (ECL1), and a long extracellular domain (ECL2). Tetraspanins are known to form specialized microdomains on the cell surface, where they interact with various proteins and signaling molecules. These interactions facilitate a wide array of cellular functions such as adhesion, motility, membrane organization, and signal transduction. CD63 serves multiple roles across different cell types: it participates in mast cell degranulation, contributes to VEGF signaling in endothelial cells, aids in leukocyte recruitment to endothelial cells, and plays a role in endosomal sorting during melanogenesis.

Background

CD63 is a member of the tetraspanin family, distinguished by its four transmembrane domains, a short extracellular domain (ECL1), and a long extracellular domain (ECL2). Tetraspanins are known to form specialized microdomains on the cell surface, where they interact with various proteins and signaling molecules. These interactions facilitate a wide array of cellular functions such as adhesion, motility, membrane organization, and signal transduction. CD63 serves multiple roles across different cell types: it participates in mast cell degranulation, contributes to VEGF signaling in endothelial cells, aids in leukocyte recruitment to endothelial cells, and plays a role in endosomal sorting during melanogenesis.

References
[1] Hemler ME, Nat Rev Mol Cell Biol. 2005 Oct;6(10):801-11. [2] Kraft S, et al. J Immunol. 2013 Sep 15;191(6):2871-8.

PVDFメンブレン孔径参考表

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

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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%