Monomeric and trimeric proteins were biotinylated and tetramerized as previously described (Bonsignori et al., 2018 (link)). Biotinylation was performed using a BirA biotin-protein ligation kit (Avidity) on proteins produced with a C-terminal avidin tag sequence. Following addition of enzyme kit reagents, proteins were agitated at 900 rpm for 5h at 30°C. Excess biotin was removed using 10kDa MW spin columns (Amicon) for monomeric proteins and 100kDA MW spin columns (Amicon) for trimeric proteins. Proteins were transferred to the spin columns after incubation and five washes were performed in PBS 1X pH7.4 (Gibco). Tetramerization of monomeric and trimeric proteins was accomplished with streptavidin (Invitrogen). A 4:1 molar ratio of protein to streptavidin was used and to maximize streptavidin site occupancy; streptavidin was added in a stepwise manner to the protein. The appropriate volume of streptavidin was added 5x every 15 min followed by agitation at 900rpm and 23°C. The final molarity of the protein was calculated based on number of moles used in the reaction as well as the total volume of protein and streptavidin combined.
Pbs 1x ph7
Manufactured by Thermo Fisher Scientific
PBS 1X pH7.4 is a widely used buffer solution that maintains a neutral pH of 7.4. It is commonly used in various laboratory applications, such as cell culture, immunoassays, and protein purification, to maintain a physiologically relevant environment for biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin, by Thermo Fisher Scientific (2 mentions)
Bira biotin protein ligation kit, by Avidity (2 mentions)
Trypan blue solution, by Thermo Fisher Scientific (1 mentions)
Trypan blue powder, by Merck Group (1 mentions)
Vi cell xr cell viability analyzer, by Beckman Coulter (1 mentions)
Countess cell counting chamber slide, by Thermo Fisher Scientific (1 mentions)
Countess automated cell counter, by Thermo Fisher Scientific (1 mentions)
U937 crl 1593.2, by American Type Culture Collection (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by GE Healthcare (1 mentions)
3 protocols using pbs 1x ph7
1
Biotinylation and Tetramerization of Proteins
2
Biotinylation and Tetramerization of Proteins
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Monomeric and trimeric proteins were biotinylated and tetramerized as previously described (Bonsignori et al., 2018 (link)). Biotinylation was performed using a BirA biotin-protein ligation kit (Avidity) on proteins produced with a C-terminal avidin tag sequence. Following addition of enzyme kit reagents, proteins were agitated at 900 rpm for 5h at 30°C. Excess biotin was removed using 10kDa MW spin columns (Amicon) for monomeric proteins and 100kDA MW spin columns (Amicon) for trimeric proteins. Proteins were transferred to the spin columns after incubation and five washes were performed in PBS 1X pH7.4 (Gibco). Tetramerization of monomeric and trimeric proteins was accomplished with streptavidin (Invitrogen). A 4:1 molar ratio of protein to streptavidin was used and to maximize streptavidin site occupancy; streptavidin was added in a stepwise manner to the protein. The appropriate volume of streptavidin was added 5x every 15 min followed by agitation at 900rpm and 23°C. The final molarity of the protein was calculated based on number of moles used in the reaction as well as the total volume of protein and streptavidin combined.
3
Comparative Cell Viability Analysis
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CHO-K1 cells (CRL-CCL 61) and U937 (CRL-1593.2) were acquired from ATCC® (Manassas, VA). RPMI 1640 medium, PBS 1X pH 7.4 and 0.4% trypan blue solution were purchased from Gibco®-Life technologies™ (New York, GI). ViaCheck™ Control beads (concentration: 1, 4 and 8 × 106 beads/mL, and viability: 0, 50, 75, 90, and 100%) were acquired from Bangs Laboratories, Inc. (Fishers, IN). Countess® Automated Cell Counter and Countess® cell counting chamber slides were acquired from Invitrogen (Carlsbad, CA). Vi-CELL® XR Cell Viability Analyzer, Vi-CELL® XR Quad Pak Reagent Kit and Coulter CC Size standard Mix kit (2, 5, 10, 20 and 43 μm latex Beads) were purchased from Beckman Coulter (Fullerton, CA). Trypan blue powder was obtained from Sigma–Aldrich (St. Louis, MO). Fetal bovine serum was acquired from PAA Laboratories (Linz, AT). DM222 A11- Liquid media were purchased from Irvine Scientific (Santa Ana, CA).
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