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CD-31 (cluster of differentiation 31) is a transmembrane protein encoded by the PECAM1 (platelet endothelial cell adhesion molecule) gene. CD-31 has essential roles in platelet biology, signal transduction, and leukocyte and endothelial cell biology.1
CD-31 can be a challenging target to work with in some applications.
CD31 is a transmembrane protein 3, 7 that requires special treatment of samples in western blot. It contains many post-transcriptional modifications, including glycosylations and phosphorylations 2, 7, 9,10,11,12, making the actual band size around 130kD, different from the predicted 79-83kD.
Add adequate protease inhibitors (or phosphatase inhibitors for proteins modified by phosphorylation) to avoid target protein degradation.
Keep samples on ice during the whole WB process.
Perform a Bradford assay, a Lowry assay or a bicinchoninic acid (BCA) assay to determine the protein concentration.
For large proteins (the MW of target protein >100 kDa), be sure to run samples in 8% or lower separating gel.
Load 20-50μg total protein per lane.
It is preferred to add SDS to a final concentration of 0.1% in the transfer buffer for large proteins.
Use Ponceau S staining to determine if the transfer is successful.
Treat samples with PNGase F or phosphatase to confirm the specificity of bands if
CD31 is widely expressed on platelets and leukocytes and is primarily concentrated at the
borders between endothelial cells. 6, 7 It can be used to help identify blood vessels and endothelial cells. Pathologists use it to identify vascular origin of tumors, but nodal sinuses may show signal as well.13 CD31 is also used to identify vascular invasion 14, 15 and assess the micro-vessel density of tumors. 16
The ideal fixation time will depend on the size of the tissue block and the type of tissue, but fixation between 18-24h is suitable for most samples.
It is recommended to optimize antibody dilution in preliminary experiments according to datasheets.
Human tonsil tissue
CD31 expresses in endothelial cells, B cells, platelets, macrophages, monocytes, NK cells, T cells. 6, 7 It is one of the biomarkers of endothelial cells. It is widely used to confirm the cell type and co-localization of proteins of interest in immunocytochemistry application.
It is recommended to fix cells in 4% PFA for 20 minutes at room temperature.
Do not over-fix your samples, which will reduce signal
It is recommended to incubate cells with 0.1% Triton-X for 5 min to detect nuclear antigen.
Use 0.3M glycine to quench autofluorescence caused by aldehydes.
Find full information on working with CD-31: