Starbright blue 520 goat anti rabbit igg

Cells were lysed with NP-40 buffer (0.2M Sodium Chloride, 0.05M Tris pH7.4, 0.5M NP-40 Alternative, 0.001M DTT, Protease Inhibitor Cocktail (Roche Complete Mini, EDTA-free tablets, 11836170001) 72 hours post transfection. The cell lysates were centrifuged at 4℃ at 1,000 rpm for 10 minutes to remove the nuclei pellet. The supernatant was transferred to a new set of a tubes and spun down at 13,000 rpm for 10 minutes at 4℃ to remove the remaining debris. The supernatant was transferred to a new set of tubes and lysed in 4X loading dye (Invitrogen, #NP0007) and boiled at 95℃ for 10 minutes. The boiled samples were resolved on a 4-12% Bis-Tris gel, transferred to a nitrocellulose membrane (Bio-Rad, 1620115), and blotted with antibodies to detect protein levels. Anti-FLAG (Sigma, F3164), and anti-p24gag (NIH-ARP, 3537) antibodies were used at 1:10,000, and Actin (Sigma, A2066), StarBright Blue 520 Goat Anti-Rabbit IgG (Bio-Rad, 12005869) and StarBright Blue 700 Goat Anti-Mouse IgG (Bio-Rad, 12005866) were used at a ratio of 1:5,000.

Mutagenesis and related PCR reactions were performed using Q5 High-Fidelity DNA Polymerase (New England Biolabs, #M0491). The mutant A3G library was constructed via multi-site saturation mutagenesis of codons 128 and 130 using NNS degenerative codons (N = A, T, C, or G, and S = G or C) within the same oligonucleotide ("128-130-NNS-R": 5'-GCTGCGAAGCGCCTCCTGGTASNNTGGSNNCCAGAAGTAGTAGAGGCGGGC-3'). An initial round of PCR was performed to produce two fragments; the first consisting of the 5' 450 bp region using the 10 units/mL Benzonase. 10 ug of lysate was resolved on a NuPAGE 4-12% Bis-Tris Protein Gel (Thermo Fisher, NP0336). Immunoblotting was performed using the primary antibodies rabbit anti-HA (Proteintech, 51064-2-AP), and mouse anti-Tubulin (Sigma-Aldrich, #T6199) at a dilution of 1:2000 and 1:5000, respectively. Secondary antibodies StarBright Blue 520 Goat Anti-Rabbit IgG (BIO-RAD, 12005869) and StarBright Blue 700 Goat Anti-Mouse IgG (BIO-RAD, 12005866) were used at a dilution of 1:10,000.