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PD-L1 (programmed cell death 1 ligand 1) is a protein encoded by the CD274 gene. PD-L1 binds to its receptor, PD-1, to modulate immune activation or inhibition and is a target in cancer immunotherapy.
PD-L1 can be a challenging target to work with in some applications.
PD-L1 is heavily glycosylated, therefore the actual band size in western blot is between 40-60kD, different from the predicted 20-33kD.
Add adequate protease inhibitors (or phosphatase inhibitors for proteins modified by phosphorylation) to avoid target protein degradation.
|Ultrasonicate samples to enrich more target proteins|
|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|
|Electrophoresis||Load 20-50μg total protein per lane.|
|Transferring||Use Ponceau S staining to determine if the transfer is successful|
|Maximizing signals||Treat samples with PNGase F to confirm the specificity of bands if necessary. 5|
|Actual band size in Western Blot is between 40-60kD.|
B-CPAP, MD-MB-231, U87-MG
PD-L1 is highly expressed in lung, ovary, and colon carcinoma and in melanomas. However, not all samples show a positive signal, and the glycosylation of PD-L1 may interfere with antibody binding.
|Sample preparation||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.|
|Under-fixation can lead to edge staining, with a strong signal on the edges of the section and no signal in the middle.|
|Over-fixation can mask the epitope. Antigen retrieval can help reverse this masking, but if the tissue has been fixed for a long period of time (ie over a weekend), there may be no signal even after antigen retrieval.|
|Antibody incubation||It is recommended to optimize antibody dilution in preliminary experiments according to datasheets.|
|Maximizing signal||Treat samples with PNGase F may increase signal intensity.5|
|To find PD-L1 expression in lung cancer tissues, please refer to the table here (PD-L1 IHC 28-8 pharmDx, Dako). For PD-L1 expression in other tumors, please refer to: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3991103/|
Human tonsil, head, and neck squamous cell carcinoma