Protein analysis experiments of recombinant viruses
Protein analysis experiments of recombinant viruses
Viruses are a class of non-cellular microorganisms with very small particles, measured in nanometers, simple structures, strict parasitism, and reproduction by replication. Viruses are microorganisms that are smaller than bacteria, have no cellular structure, and can only proliferate in cells. They are composed of proteins and nucleic acids. Most have to be observed with an electron microscope.
Operation method
basic program
Materials and Instruments
Sf Cells Move 1. 2. 5x108 Sf9 cells are inoculated into 25 cm2 flasks containing 5 ml complete culture medium/10% fetal bovine serum and incubated at 27°C for ≥2 h. 0.5 ml of the 1 ml virus reservoir containing serum-free complete culture medium and recombinant etch was added to each flask. 0.5 ml of the remaining recombinant virus was stored at 4°C to be used in etching assays. 2. The flasks were incubated at 27°C for 4-5 days, checking for signs of infection every day. For more product details, please visit Aladdin Scientific website.
Fetal Bovine Serum PBS SDS Protease Inhibitors Lysis Buffer
Culture flasks Incubators Centrifuges Rotors Water baths
2. incubate the flasks at 27°C for 4-5 days, checking for signs of infection every day. after 4-5 days, transfer the culture to a 15 ml centrifuge tube and harvest the virus.3. Centrifuge at 1,000 g for 10 min at 4°C and transfer the supernatant containing the amplified virus to a sterile 15 ml polypropylene centrifuge tube.4. Inoculate 2. 5x106 Sf9 cells in 25 cm2 flasks containing 5 ml complete culture medium/10% fetal bovine serum. Prepare one culture flask for each - virus reservoir to be tested and another one as a non-infected control. incubate at 27°C for ≥2 h to allow the cells to adhere to the wall. Discard the cell culture medium and add 1.5 ml of amplified virus reservoir to the culture flasks and incubate at 27°C for 2 days. 5.
5. Gently remove the cells from the tower culture flasks to harvest them and transfer the cells and culture solution to 15 ml polypropylene centrifuge tubes.6. Centrifuge the cells at 1,000 g for 10 min at 4°C. If the target protein is a secreted protein, transfer the culture supernatant to a new tube and proceed to step 8. If the protein to be studied is intracellular, discard the supernatant, gently resuspend the cell pellet in PBS, repeat centrifugation and discard the supernatant.
7. Add 500 ul of 1xSDS sample buffer directly to the cell pellet, boil the pellet, sonicate the sample if it is too viscous due to the presence of DNA, sonicate the sample and continue sonication until the viscosity is removed, then proceed to step 9. Another alternative is to lysed the cell pellet with 0.5 ml of appropriate lysis buffer supplemented with protease inhibitors, microcentrifuge for 10 minutes at 4°C to clarify the lysate, and transfer the supernatant to a new tube. The supernatant was transferred to a new tube. Add 0.1 ml of each lysate to 100 ul of 2xSDS sample buffer and boil in a boiling water bath for 3 min. Freeze the remaining lysate at -80°C for several months. 8.
8. Determine the concentration of secreted proteins in the culture supernatant (step 6) or intracellular proteins in the cell lysate (step 7) using Bradford's method.
9. analyze the proteins in each sample by one of the following methods:(1) Immunoblotting: on a one-way SDS-polyacrylamide gel, add 20 to 40 ug of total cellular protein per lane. Remember to include non-infected controls.
(2) Caulmers Brilliant Blue Staining: On a unidirectional SDS-polyacrylamide gel, add 20~40 ug total cell protein per lane. If the recombinant virus is impure, use this assay only if the recombinant protein yield in infected cells is high.
(3) Functional analysis: e.g., mobility-shifted DNA binding assay, in vitro kinase assay (for protein kinases), nucleoside binding assay (for nucleoside-bound proteins), thymidine doping assay (for growth factor proteins), or any of the typical assays used for detection of the target protein.
(4) Metabolic labeling of recombinant proteins.
10. interpret the results to identify which of the putative recombinant eclipses is the recombinant that produces the desired protein, which will be eclipsed and purified from any wild-type viral contamination. Prepare a large reservoir of viral stock and determine the titer of the recombinant virus.