Western blotting and reporter assay were performed as previously described [12 (link)–14 (link), 45 (link)–47 (link), 52 (link)]. For the Western blotting of virus particles, 340 μl of the culture supernatant was ultracentrifuged at 100,000×g for 1 h at 4 °C using a TL-100 instrument (Beckman), and the pellet was lysed with 1 × SDS buffer. For the Western blotting of transfected cells, the cells were lysed with RIPA buffer (50 mM Tris–HCl buffer [pH 7.6], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitor cocktail (Roche). The following antibodies for Western blotting: anti-His polyclonal antibody (OGHis; Medical and Biological Laboratories), anti-HA antibody (3F10; Roche), anti-FIV p24 Capsid antibody (PAK3-2C1; Santa Cruz Biotechnology); anti-alpha-tubulin (TUBA) antibody (DM1A; Sigma), and anti-VSVg antibody (P5DA; Roche). For FIV reporter assays, HEK293T cells were used for the target of infection. Ten microliter of the culture supernatant of transfected cells was inoculated into HEK293T cells in a 96-well plate (Nunc), and the firefly luciferase activity was measured by using the BrillianStar-LT assay system (Toyo-b-net) and the 2030 ARVO X multilabel counter instrument (PerkinElmer) according to the manufacturers’ procedures.
Tl 100 instrument
Manufactured by Beckman Coulter
The TL-100 instrument is a high-speed refrigerated tabletop centrifuge designed for a variety of laboratory applications. It features a brushless motor and a wide range of rotor options to accommodate different sample sizes and types. The TL-100 provides precise control of speed, time, and temperature to ensure consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (5 mentions)
Anti ha antibody 3f10, by Roche (3 mentions)
96 well plate, by Thermo Fisher Scientific (2 mentions)
Anti his polyclonal antibody oghis, by Medical & Biological Laboratories (2 mentions)
Anti fiv p24 capsid antibody pak3 2c1, by Santa Cruz Biotechnology (2 mentions)
Anti vsvg antibody p5da, by Roche (2 mentions)
Anti alpha tubulin tuba antibody dm1a, by Merck Group (1 mentions)
Anti ha antibody, by Roche (1 mentions)
2030 arvo x multilabel counter instrument, by PerkinElmer (1 mentions)
Anti c myc antibody c3956, by Merck Group (1 mentions)
0.45 μm pore size filter, by Merck Group (1 mentions)
Anti ha clone 3f10, by Roche (1 mentions)
Image lab v5, by Bio-Rad (1 mentions)
Mouse anti flag monoclonal antibody clone m2, by Merck Group (1 mentions)
6 protocols using tl 100 instrument
1
Western Blotting and Reporter Assay for Virus Particles
2
Western Blotting of Virus Particles and Transfected Cells
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Western blotting was performed as previously described (22 (link), 24 (link), 36 (link), 62 (link)). For the Western blotting of virus particles, 340 μl of the culture supernatant was ultracentrifuged at 100,000 × g for 1 h at 4°C using a TL-100 instrument (Beckman), and the pellet was lysed with 1× SDS buffer. For the Western blotting of transfected cells, the cells were lysed with RIPA buffer (50 mM Tris-HCl buffer [pH 7.6], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitor cocktail (Roche). The following antibodies were used for Western blotting: anti-His polyclonal antibody (OGHis; Medical and Biological Laboratories), anti-HA antibody (3F10; Roche), anti-FIV p24 capsid antibody (PAK3-2C1; Santa Cruz Biotechnology); and anti-alpha-tubulin (TUBA) antibody (DM1A; Sigma).
3
Immunoblot analysis of viral and cellular proteins
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SDS-PAGE/immunoblot analysis was performed as previously described (29 (link), 30 (link)) by using the following antibodies: anti-HA antibody (3F10; Roche); anti-Flag antibody (OctA; Santa Cruz); anti-c-Myc antibody (C3956; Sigma-Aldrich); anti-FIV p27 antibody (PAK3-2C1; Santa Cruz); anti-VSVg antibody (P5DA; Roche); anti-FeLV p24 antibody (PF12J-10A; Santa Cruz); anti-RD-114 virus capsid (CA) antibody (37 (link)); and anti-α-tubulin (TUBA) antibody (DM1A [Sigma-Aldrich] or B-5-1-2 [Covance]). For the virus, 500 μl of the virus solution was ultracentrifuged at 100,000 × g for 1 h at 4°C using a TL-100 instrument (Beckman), and the pellet was lysed with 1× SDS buffer. For the transfected cells, the cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-HCl buffer [pH 7.6], 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) with protease inhibitor cocktail (Roche).
To calculate the percentage of feline A3Z3 degradation (see percent degradation data inFig. 5C and D ), the band intensities of the immunoblots of feline A3Z3-HA and TUBA were quantified by using Image J software (http://imagej.nih.gov/ij/ ), and the following formula was used: percent degradation = 100 − {[A3Z3-HA (+Vif)]/[TUBA (+Vif)]}/{[A3Z3-HA (−Vif)]/[TUBA (−Vif)]} × 100.
To calculate the percentage of feline A3Z3 degradation (see percent degradation data in
4
Viral Infectivity Measurement and Protein Analysis
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The culture supernatant harvested at 48 hours post-transfection was centrifuged and then filtered through a 0.45-μm-pore-size filter (Milipore) to produce virus solution. The infectivity of virus solution was measured by TZM-bl assay as previously described18 (link)19 (link)58 (link). Briefly, 10 μl of the virus solution was inoculated into TZM-bl cells in 96-well plate (Nunc), and the β-galactosidase activity was measured by using the Galacto-Star mammalian reporter gene assay system (Roche) and a 2030 ALBO X multilabel counter instrument (PerkinElmer) according to the manufacturers' procedure. Western blotting was performed as previously described18 (link)19 (link)58 (link) by using the following antibodies: anti-p24 polyclonal antibody (ViroStat), anti-KGC antibody (clone 21B10; Medical and Biological Laboratories, Inc.), anti-HA antibody (3F10; Roche), and anti-α-Tubulin (TUBA) antibody (DM1A; Sigma). For the virus solution, 500 μl of the virus solution was ultracentrifuged at 100,000 × g for 1 hour at 4°C using a TL-100 instrument (Beckman). The pellet was lysed with 1 × SDS buffer, and the lysate was used for SDS-PAGE/Western blotting. For the transfected cells, the cells were lysed with 1 × SDS buffer, and the lysate was used for SDS-PAGE/Western blotting.
5
Western Blotting of Viral Proteins
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Western blotting was performed as previously described (22 (link), 60 (link), 68 (link)) using the following antibodies: anti-HA (clone 3F10; Roche), anti-Flag (rabbit polyclone OctA; Santa Cruz Biotechnology), anti-His (clone OGHis; Medical and Biological Laboratories), anti-FIV p27 (clone PAK3-2C1; Santa Cruz Biotechnology), anti-vesicular stomatitis virus G glycoprotein (anti-VSVg; clone P5DA; Roche), and anti-α-tubulin (TUBA; clone DM1A; Sigma-Aldrich). Five hundred microliters of viral supernatant was ultracentrifuged at 100,000 × g for 1 h at 4°C using a TL-100 instrument (Beckman), and the pellet was lysed with 1× SDS buffer. Transfected cells were lysed with radioimmunoprecipitation assay (RIPA) buffer containing a protease inhibitor cocktail (Roche). To quantify the amount of feline A3Z2Z3 and cleaved A3Z3 in the released viral particle (Fig. 7F ), the band intensities of feline A3Z2Z3 and cleaved A3Z3 in the supernatant blot (a representative blot is shown in Fig. 7D ) were quantified using Image Lab (v5.2) software (Bio-Rad).
6
Western Blotting of Viral Particles
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Western blotting was performed as described [30 (link), 36 (link)] using the following antibodies: anti-HA mouse monoclonal antibody (clone 3F10; Roche), anti-Flag mouse monoclonal antibody (clone M2; Sigma-Aldrich), anti-p24 goat antiserum (ViroStat), anti-alpha-tubulin mouse monoclonal antibody (TUBA; clone DM1A; Sigma-Aldrich); anti-hA3G rabbit antiserum (the NIH AIDS Research and Reference Reagent Program catalog number #10201); and anti-CBF-β mouse monoclonal antibody (sc-56751; Santa Cruz). For Western blotting of viral particles, 370 μl of viral supernatant was ultracentrifuged at 100,000 × g for 1 h at 4°C using a TL-100 instrument (Beckman), and the pellet was lysed with 1 × SDS buffer. Transfected cells were lysed with RIPA buffer (25 mM HEPES [pH 7.4], 50 mM NaCl, 1 mM MgCl2, 50 μM ZnCl2, 10% glycerol, 1% Triton X-100) containing a protease inhibitor cocktail (Roche).
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