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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.
Overview
Protein function, expression, and isoforms
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.
Sample preparation | 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. | |
Controls | Positive: B-CPAP, MD-MB-231, U87-MG Negative: A549 SW480 |
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/ | |
Controls | Positive: Human tonsil, head, and neck squamous cell carcinoma Placenta tissues Negative: MCF-7 5 |