Difference between revisions of "Part:BBa K2976008"
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<p>We transfected Plasmid (anti-PD-L1-lamp2 fusion protein-hsa-let-7f) into RAW264.7 cells to express the specific exosomes, and harvest the exosomes by way of exosome extracting kit. RAW264.7 cells were transfected with plasmids for 24 hours, and the agonist Pam3Cys was added after 24 hours of transfection. Exosomes were harvested after another incubation of 24 hours. The purified exosomes were observed under electron microscopy as shown in Figure 1. Then we immediately extracting the exosome protein as well as the overall protein of cells. Through Western Blot, we successfully obtain the signal of HA tag and the marker protein of exosome CD63 from the exosome sample, but there is no signal of HA tag from that of cells, this illustrates that the HA tag was indeed expressed and specifically expressed in exosomes. (Fig.2). | <p>We transfected Plasmid (anti-PD-L1-lamp2 fusion protein-hsa-let-7f) into RAW264.7 cells to express the specific exosomes, and harvest the exosomes by way of exosome extracting kit. RAW264.7 cells were transfected with plasmids for 24 hours, and the agonist Pam3Cys was added after 24 hours of transfection. Exosomes were harvested after another incubation of 24 hours. The purified exosomes were observed under electron microscopy as shown in Figure 1. Then we immediately extracting the exosome protein as well as the overall protein of cells. Through Western Blot, we successfully obtain the signal of HA tag and the marker protein of exosome CD63 from the exosome sample, but there is no signal of HA tag from that of cells, this illustrates that the HA tag was indeed expressed and specifically expressed in exosomes. (Fig.2). | ||
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| − | [[File:T--CPUCHINA--dianjing.jpg|250px|middle|Figure 1: Electron microscopy of exosomes.]] | + | [[File:T--CPUCHINA--dianjing.jpg|250px|thumb|middle|Figure 1: Electron microscopy of exosomes.]] |
| − | [[File:T--CPUCHINA--HAtag.jpg|200px|middle|Figure 2: Western blot result of exosome protein.]] | + | [[File:T--CPUCHINA--HAtag.jpg|200px|thumb|middle|Figure 2: Western blot result of exosome protein.]] |
Revision as of 14:57, 21 October 2019
HA tag
Usage and Biology
Protein tags are protein or peptide sequences located either on the C- or N- terminal of the target protein, which facilitates the following characteristics: solubility, detection, purification, localization and expression. The HA tag (YPYDVPDYA) is derived from an epitope of the influenza hemagglutinin protein which has been used extensively as a general epitope tag in expression vectors due to its small size and low influence on the tagged protein’s biochemical properties. In 2019 CPU_CHINA project, we insert HA tag into the fusion proteins on the membrane of exosomes for the immunoblotting assay.
Characteristics
We transfected Plasmid (anti-PD-L1-lamp2 fusion protein-hsa-let-7f) into RAW264.7 cells to express the specific exosomes, and harvest the exosomes by way of exosome extracting kit. RAW264.7 cells were transfected with plasmids for 24 hours, and the agonist Pam3Cys was added after 24 hours of transfection. Exosomes were harvested after another incubation of 24 hours. The purified exosomes were observed under electron microscopy as shown in Figure 1. Then we immediately extracting the exosome protein as well as the overall protein of cells. Through Western Blot, we successfully obtain the signal of HA tag and the marker protein of exosome CD63 from the exosome sample, but there is no signal of HA tag from that of cells, this illustrates that the HA tag was indeed expressed and specifically expressed in exosomes. (Fig.2).
