Western blot coomassie blue stain

CD31 target tips

​CD31 (cluster of differentiation 31) is a transmembrane protein encoded by the PECAM1 (platelet endothelial cell adhesion molecule) gene. CD31 has essential roles in platelet biology, signal transduction, as well as leukocyte and endothelial cell biology.1 

Overview​​

​Sample preparation

Electrophoresis

Transferring

Recommended controls

​Sample fixation

Antibody incubation 

Recommended controls

​Sample fixation

Antibody incubation

Recommended controls

​

​​

​​Protein function, expressions, and isoforms

Protein function

  • CD31 is a cell adhesion molecule that is required for leukocyte transendothelial migration under most inflammatory conditions. 2 3
  • Heterophilic interaction with CD177 plays a role in the transendothelial migration of neutrophils. 3 
  • ​Homophilic ligation of PECAM1 prevents macrophage-mediated phagocytosis of neighboring viable leukocytes by transmitting a detachment signal. 4
  • Promotes macrophage-mediated phagocytosis of apoptotic leukocytes by tethering them to the phagocytic cells; PECAM1-mediated detachment signal appears to be disabled in apoptotic leukocytes. 4
  • Modulates bradykinin receptor BDKRB2 activation. Regulates bradykinin- and hyperosmotic shock-induced ERK1/2 activation in endothelial cells. 5

​Expression

  • Expressed on platelets, leukocytes, neutrophils 3, human umbilical vein endothelial cells (HUVECs). 2 3
  • CD31 is primarily concentrated at the borders between endothelial cells. 6 7
  • Isoform long predominates in all tissues examined. 8
​​Isoforms
  • Human: Isoform 1-6: 79-83kD (predicted)
  • Mouse: Isoform 1-4: 69.8-81.3kD (predicted)


​

Western blot tips

CD31 is a transmembrane protein 3, 7 that requires special treatment of samples in western blot. It contains many post-translational modifications, including glycosylations and phosphorylations 2, 7, 9,10,11,12,

  • The actual band size is around 130kD, different from the predicted 79-83kD
  • To confirm the band is indeed CD31, you can treat with phosphatase and PGNase F if necessary. This treatment will break up these post-translational modifications.
Sample preparation

Back to top

Electrophoresis

  • Run samples in 8% or lower separating gel.
  • Load 20 – 50 μg total protein per lane.

Back to top

Transferring 
  • We recommend adding SDS to a final concentration of 0.1% in the transfer buffer.
  • Determine whether the transfer is successful by visualization of proteins in membranes using Ponceau S.

Back to top

Recommended controls

Positive: HUVEC and Jurkat cell lysates (ab7899); Human spleen and kidney tissue lysate.

Negative: NIH/3T3 whole cell lysate (ab7179)

Back to top

​​


​​IHC tips

Immunostaining CD31 can be used to help identify blood vessels and endothelial cells, where this protein tends to be most concentrated. Pathologists use it to identify vascular origin of tumors, although nodal sinuses may also show a signal 13. CD31 is also used to identify vascular invasion 14, 15 and assess the micro-vessel density of tumors 16​.​

Sample fixation
  • The ideal fixation time will depend on the size of the tissue block and type of tissue, but  18–24 hours is suitable for most samples.

​Back to top

Antibody incubation
  • It is recommended to optimize antibody dilution in preliminary experiments according to datasheets.

Back to top

Recommended controls

Positive: Human tonsil tissue

Back to top


​

ICC tips


CD31 can be used as a biomarker for endothelial cells. It is widely used to confirm the cell type and co-localization of proteins of interest in immunocytochemistry.

Sample fixation
  • We recommend fixing the cells in 4% PFA for 20 minutes at room temperature.
  • Be sure not to over-fix your samples, as this will reduce the signal.

Back to top

Antibody incubation
  • If using fluorescent detection, add 0.3M glycine to the sample. This will quench autofluorescence caused by aldehydes.

Back to top

Recommended controls

Positive: HUVEC cells

Back to top 

​​

References

  1. Lertkiatmongkol, P., Liao, D., Mei, H., Hu, Y., Newman, P.J. Endothelial functions of platelet/endothelial cell adhesion molecule-1 (CD31). Curr Opin Hematol. 23, 253-259 (2016).  
  2. Dasgupta, B., Dufour, E., Mamdouh, Z., Muller, W.A. A novel and critical role for tyrosine 663 in platelet endothelial cell adhesion molecule-1 trafficking and transendothelial migration. J Immunol. 182, 5041-51 (2009)
  3. Sachs, U.J., Andrei-Selmer, C.L., Maniar, A. et al. The neutrophil-specific antigen CD177 is a counter-receptor for platelet endothelial cell adhesion molecule-1 (CD31). J Biol Chem. 282, 23603-12 (2007). 
  4. Brown, S., Heinisch, I., Ross, E., Shaw, K., Buckley, C.D., Savill, J. Apoptosis disables CD31-mediated cell detachment from phagocytes promoting binding and engulfment. Nature. 418, 200-3 (2002) 
  5. Yeh, J.C., Otte, L.A., Frangos, J.A. Regulation of G protein-coupled receptor activities by the platelet-endothelial cell adhesion molecule, PECAM-1. Biochemistry. 26, 9029-39 (2008)
  6. Bergom, C., Paddock, C., Gao, C., Holyst, T., Newman, D.K., Newman P.J. An alternatively spliced isoform of PECAM-1 is expressed at high levels in human and murine tissues, and suggests a novel role for the C-terminus of PECAM-1 in cytoprotective signaling. J Cell Sci. 121, 1235-42 (2008)
  7. Paddock, C., Lytle, B.L., Peterson, F.C., Holyst, T., Newman, P.J., Volkman, B.F., Newman, D.K. Residues within a lipid-associated segment of the PECAM-1 cytoplasmic domain are susceptible to inducible, sequential phosphorylation. Blood. 117, 6012-23 (2011)
  8. Wang, Y., Su, X., Sorenson, C.M., SheibaniN. Tissue-specific distributions of alternatively spliced human PECAM-1 isoforms. Am J Physiol Heart Circ Physiol. 284, H1008-17 (2003) 
  9. Paddock, C., Zhou, D., Lertkiatmongkol, P., Newman, P.J., Zhu, J. Structural basis for PECAM-1 homophilic binding. Blood. 127, 1052-61 (2016)
  10. Wollscheid, B., Bausch-Fluck, D., Henderson, C., O'Brien, R., Bibel, M., Schiess, R., Aebersold, R., Watts, J.D. Mass-spectrometric identification and relative quantification of N-linked cell surface glycoproteins. Nat Biotechnol. 27, 378-86 (2009)
  11. Chen, R., Jiang, X., Sun, D., Han, G., Wang, F., Ye, M., Wang, L., Zou, H. Glycoproteomics analysis of human liver tissue by combination of multiple enzyme digestion and hydrazide chemistry. J Proteome Res. 8, 651-61 (2009) 
  12. Famiglietti, J., Sun, J., DeLisser, H.M., Albelda, S.M. Tyrosine residue in exon 14 of the cytoplasmic domain of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) regulates ligand binding specificity. J Cell Biol. 138, 1425-35 (1997)
  13. Hattori, H. Caution should be taken in using CD31 for distinguishing the vasculature of lymph nodes. J Clin Pathol. 56, 638-9 (2003)
  14. Alexander-Sefre, F., Singh, N., Ayhan, A., Salveson, H.B., Wilbanks, G., Jacobs, I.J. Detection of tumour lymphovascular space invasion using dual cytokeratin and CD31 immunohistochemistry. J Clin Pathol. 56, 786-8 (2003) 
  15. Kurtz, K.A., Hoffman, H.T., Zimmerman, M.B., Robinson, R.A. Perineural and vascular invasion in oral cavity squamous carcinoma: increased incidence on re-review of slides and by using immunohistochemical enhancement. Arch Pathol Lab Med. 129, 354-9. (2005)
  16. El-Gohary, Y.M., Metwally, G., Saad, R.S., Robinson, M.J., Mesko, T., Poppiti, R.J. Prognostic significance of intratumoral and peritumoral lymphatic density and blood vessel density in invasive breast carcinomas. Am J Clin Pathol. 129, 578-86. (2008)

​

Download these tips as a PDF

Find full information on working with CD-31:
In English (link to pdf)
In Mandarin (Link to pdf)

​