Vesicular stomatitis virus G protein binding assay
Vesicular stomatitis virus G protein binding assay
The capsid fusion G protein of vesicular stomatitis virus (VSV-G) can be used to prepare pseudotyped retroviruses and pseudolentiviruses, and this glycoprotein can also be used alone as an effective gene transfer vector. Cells transfected with the VSV-G expression plasmid in the absence of other viral components secreted the VSV-G protein into the culture medium and could be partially purified by ultracentrifugation in the sucrose layer. Author: T. Friedman et al, Translated by W. Qin et al. This experiment is from "Gene Transfer".
Operation method
Preparation of VSV-G conjugate and use for gene delivery Move Preparation of VSV-G conjugate and use for gene delivery Materials reagents B C A Protein Quantification Kit (Pierce, Rockford, Illinois) Cell Culture Media H E K 293 Cells (A T C C ) Phosphate buffer (P B S ) Plasmid Reporter gene expression plasmids (e.g., E G F P -N l , B D Biosciences, Palo Alto, California) V S V - G expression plasmid p H C M V - G (Yee et al. 1994a , b) Polybrene (4m g / m l reservoir) S D S - P A G E and silver staining reagents Western blot analytical reagents Receptor Cells Sucrose (20%) / P B S Instrument Centrifuge with S W 28 rotor and centrifuge tubes (Beckman) SDS-PAGE and silver-staining equipment Western blot analyzer Microporous Filter (○.45um) Cell plates (6 wells) Tissue Culture Dish (IOcm) 37°C water bath Methods Preparation VSV-G Any transfection method can be used to transhumanize VSV-G expression plasmids into cells to express VSV-G protein. We generally chose the HEK293 cell line because it can be transfected by any method, including CaP 〇 4_D N A co-precipitation, and all have high transfection (Yeeetal.1994a, b). 1- Before transfection for 24 h , H E K 293 cells were grown overnight in I O c m dishes inoculated at a cell density of approximately 70% with a suitable culture medium. At the time of transfection, the cell density should be 90% to 100%. 2- Transfect 100~200ug of PHCMV-G plasmid into HEK293 cells by standard CaPCVDNA co-precipitation method. Because HEK293 cells are sensitive to the toxicity of CaPO4 precipitation, the transfection time should be shorter than 8 hours. 3. The next day, replace the fresh culture medium. Because of the fusion effect of V S V - G, multinucleosomes may form, but should not be apparent at this time. Continue the culture overnight. 4 . At this point the polysomes should be very visible. Collect the culture medium, filter through a 0.45um membrane and store at 70°C. Add fresh culture medium to the cells and continue to incubate overnight. Add fresh culture medium to the cells and continue to incubate overnight. 5. Many cells may float, collect culture medium and filter as in step 4. Store at 70°C or purify. Partial purification of VSV-G (simple method) 6. Partial purification of V S V -G , with the following steps: b . Remove insoluble debris by centrifugation at low speed (e.g., 3000 r/m i n at 4°C for 100 m i n ). Add the supernatant to 20% sucrose/PBS (5 ml), place in a centrifuge tube with a W 28 rotor, cover and fill the tube with PBS, and centrifuge at 25,000 r/m i n for 6 h or overnight. Discard the supernatant and resuspend the precipitate with 100-20(V 1P B S). c. Measure the concentration of VSV-G protein with BCA Protein Quantification Kit. Protein purity was estimated by SD& PAGE, silver staining and Western blot analysis using anti-VSV-G monoclonal antibody P5D4. Transfection with VSV-G 7 . 24 h before transfection, inoculate the cells to be transfected with appropriate medium in a 6-well plate and incubate overnight. 8 - Reduce the culture medium in the wells to lm l per well, add POlybr e n e to the culture medium (4ug/ml). In a small centrifuge tube, mix 2 to 5 ug of plasmid DNA (e.g. PEGFP-N1) with 10ul of approximately 1ug of VSV-G solution. Add it quickly to the cells. 9-Mix well and incubate in the incubator for 2~6 h. Then add Im l of fresh medium to each well and continue incubation. If there are more fragments, wash the cells and add 2 ml of fresh medium. 10- Measure the transgene expression in 1~3d to evaluate the transfection efficiency of the cells. For more product details, please visit Aladdin Scientific website.
Anti-V S V - G monoclonal antibody P 5D 4 ( Sigma-Aldrich)
a. Thaw and mix the culture medium harvested from transfection 2d and 3d in a 37°C water bath. Transfer the culture solution to a BECK-MANSW28 rotor-specific centrifuge tube and centrifuge at 25,000 r/min for 2 h at 4°C. Discard the supernatant. Resuspend the starch with 5-IOml of PBS. If the medium is more than 200 ml, the VSV-G can be collected by overnight centrifugation with a large capacity rotor at 6000 to 7000 r/m i n .
Alternatively, plasmid DNA can be added directly to cells in Im l culture medium and incubated for 15m in, then polybrene and VSV-G are added.