Figure 1: CD9 target protein structure.

CD9 Target Introduction

Protein Function

• CD9 is a transmembrane member of the tetraspanin family. It is also an integrin-associated membrane protein that regulates sperm-egg fusion, platelet activation and aggregation, and cell adhesion.
• In macrophages, CD9 function is associated with CD81 and β-1 and β-2 integrins, and it prevents macrophage fusion into multinucleated giant cells, which are specialized in engulfing complement-opsonized particles.
• CD9 is involved in platelet activation and aggregation. It also participates in cell adhesion, cell motility, and tumor metastasis.
• CD9 was initially thought to be a specific exosome marker; however, it is now considered an anti-inflammatory marker in monocytes and macrophages.

Protein Expression

• CD9 is expressed in various hematopoietic and non-hematopoietic cells, including stromal cells, megakaryocytes, platelets, B and T lymphocytes, dendritic cells, endothelial cells, mast cells, eosinophils, and basophils.

Protein Localization

• Extracellular or extravesicular, cell membrane.

Figure 2: ICC experimental results of CD9 target, using the Anti-CD9 antibody [EPR23105-121] product (ab236630). Green: CD9, Red: alpha Tubulin, Blue: DAPI.

Isoforms & Post-translational modifications

• Human (P21926): 25.4 kDa (predicted)
• Mouse (P40240): 25.2 kDa (predicted)
• Rat (P40241): 25.2 kDa (predicted)
• Palmitoylation
• CD9 protein exists in three forms, with molecular weights ranging from 22 to 27 kDa, and can covalently bind with fatty acids.
• Disulfide bonds, glycoprotein, lipoprotein, palmitic acid ester

WB experiment tips

Precautions

• CD9 is expressed in some tissues and cell lines, and its expression level also varies in different tissues and cell lines. We recommend setting a positive control.
• CD9 is a multi-pass transmembrane protein, and it is strongly recommended not to boil the sample when extracting the protein to prevent protein aggregation; for cells with low expression levels, target proteins can be enriched by extracting membrane proteins.
• When detecting CD9 in exosomes, the protein abundance of CD9 is very important. There may be no signal if the extracted exosomes have a low CD9 content. For more exosome extraction protocols and research guidelines, please refer to references [1,2].
• CD9 is a protein with a relatively small molecular weight, and experiments using the workflow for small molecular weight proteins during electrophoresis and membrane transfer are recommended.
• The actual detected molecular size may not match the prediction. The predicted band size is approximately 25 kDa, while the detected band size is approximately 22 kDa.

Positive control

• HeLa and HCT116 whole cell lysate
• Mouse heart and kidney tissue lysate

Negative control (no expression or weak expression)

• Raji whole cell lysat
• Human CD9 knockout HeLa cell line (ab255375)

Example of results

Figure 3: WB-Anti-CD9 antibody [EPR23105-121] product (ab236630).

Primary antibody: Anti-CD9 antibody [EPR23105-121] was used at a dilution of 1/1000.
Lane 1: Human amygdala lysate; Lane 2: Human colon lysate.
Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used at a dilution of 1/100000.

Predicted band size: 25 kDa
Detected band size: 22 kDa

Figure 4: WB-Anti-CD9 antibody [EPR23105-121] product (ab236630).
Primary antibody: Anti-CD9 antibody [EPR23105-121] was used at a dilution of 1/1000.

Lane 1: HCT116 whole cell lysate;
Lane 2: Raji whole cell lysate.

Secondary antibody: Goat Anti-Rabbit IgG H&L (HRP) (ab97051) was used at a dilution of 1/100000.
Predicted band size: 25 kDa
Detected band size: 22 kDa

Key control points

In the experiment, special attention should be given to key control points in addition to routine issues:

Sample preparation:

1. Add a complex protease inhibitor to avoid degradation of the target protein.
2. Keep the sample on ice throughout the sample preparation process.
3. For membrane proteins, we strongly recommend not boiling the sample.
4. Determine the total protein concentration of the sample through Bradford analysis, Lowry analysis, or BCA analysis.
5. We recommend using positive and negative controls.

Electrophoresis:

1. Load at least 20 μg total protein for electrophoresis.
2. For target proteins with smaller molecular weights, use a higher concentration separation gel, such as a 15% separation gel, for electrophoresis.

Transfer:

1. For target proteins with smaller molecular weights, it is recommended to use a 0.22 μm PVDF membrane.
2. For target proteins with smaller molecular weights, it is recommended to use 20% methanol in the transfer buffer.
3. We strongly recommend using Ponceau S staining after transfer to confirm the success of the transfer.
4. We recommend not cutting the membrane.

Antibody incubation:

1. We recommend using fresh antibodies and not reusing antibodies.

References

1. Emma J K Kowal, Dmitry Ter-Ovanesyan, Aviv Regev, etc. Extracellular Vesicle Isolation and Analysis by Western Blotting. Methods Mol Biol. 2017;1660:143-152. doi: 10.1007/978-1-4939-7253-1_12
2. Clotilde Théry, Kenneth W Witwer, Elena Aikawa, etc. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018 Nov 23;7(1):1535750. doi: 10.1080/20013078.2018.1535750.
3. Brosseau C, Colas L, Magnan A, Brouard S. CD9 Tetraspanin: A New Pathway for the Regulation of Inflammation? Front Immunol. 2018, 9:2316. doi: 10.3389/fimmu.2018.02316.
4. Takeda Y, Tachibana I, Miyado K et al. Tetraspanins CD9 and CD81 function to prevent the fusion of mononuclear phagocytes. J Cell Biol. 2003,161(5):945-56. doi: 10.1083/jcb.200212031.
5. Nakazawa Y, Sato S, Naito M, Kato Y, Mishima K, Arai H, Tsuruo T, Fujita N. Tetraspanin family member CD9 inhibits Aggrus/podoplanin-induced platelet aggregation and suppresses pulmonary metastasis. Blood. 2008,112(5):1730-9. doi: 10.1182/blood-2007-11-124693.
6. Ikeyama S, Koyama M, Yamaoko M, Sasada R, Miyake M. Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA. J Exp Med. 1993, 177(5):1231-7. doi: 10.1084/jem.177.5.1231.
7. Masellis-Smith A, Shaw AR. CD9-regulated adhesion. Anti-CD9 monoclonal antibody induce pre-B cell adhesion to bone marrow fibroblasts through de novo recognition of fibronectin. J Immunol. 1994,152(6):2768-77.
8. Charrin S, Manié S, Oualid M, Billard M, Boucheix C, Rubinstein E. Differential stability of tetraspanin/tetraspanin interactions: role of palmitoylation. FEBS Lett. 2002 Apr 10;516(1-3):139-44. doi: 10.1016/s0014-5793(02)02522-x.
9. Israels SJ, McMillan-Ward EM. Palmitoylation supports the association of tetraspanin CD63 with CD9 and integrin alphaIIbbeta3 in activated platelets. Thromb Res. 2010, 125(2):152-8. doi: 10.1016/j.thromres.2009.07.005.