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.
Anti alpha tubulin tuba antibody dm1a
Manufactured by Merck Group
The Anti-alpha-tubulin (TUBA) antibody (DM1A) is a laboratory reagent used for the detection and analysis of alpha-tubulin, a key component of the cytoskeleton in eukaryotic cells. This antibody can be utilized in various immunoassay techniques, such as Western blotting and immunohistochemistry, to study the expression and localization of alpha-tubulin within biological samples.
Automatically generated - may contain errors
Lab products found in correlation
96 well plate, by Thermo Fisher Scientific (3 mentions)
2030 arvo x multilabel counter instrument, by PerkinElmer (3 mentions)
Anti ha antibody 3f10, by Roche (3 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Galacto star mammalian reporter gene assay system, by Thermo Fisher Scientific (2 mentions)
Tl 100 instrument, by Beckman Coulter (1 mentions)
Anti his polyclonal antibody oghis, by Medical & Biological Laboratories (1 mentions)
Anti ha antibody, by Roche (1 mentions)
Anti fiv p24 capsid antibody pak3 2c1, by Santa Cruz Biotechnology (1 mentions)
Anti vsvg antibody p5da, by Roche (1 mentions)
0.45 μm filter, by Merck Group (1 mentions)
Ac 15, by Merck Group (1 mentions)
4 protocols using anti alpha tubulin tuba antibody dm1a
1
Western Blotting and Reporter Assay for Virus Particles
2
Virus Infectivity Quantification and Protein Analysis
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The culture supernatant harvested at 48 hours post-transfection was centrifuged and filtered through a 0.45-μm filter (Millipore) to produce virus suspensions. The infectivity of the virus suspensions was measured by a TZM-bl assay as previously described50 (link)51 (link). Briefly, 100 μl of the virus solution were inoculated onto TZM-bl cells in a 96-well plate (Nunc), and the β-galactosidase activity was measured using the Galacto-Star mammalian reporter gene assay system (Applied Biosystems) and a 2030 ARVO X multilabel counter instrument (PerkinElmer) according to the manufacturers’ instructions. Western blotting was performed as previously described37 (link)50 (link)51 (link) using the following antibodies: anti-CA polyclonal antibody (ViroStat), anti-flag polyclonal antibody (OctA; Santa Cruz Biotechnology), anti-HA antibody (3F10; Roche), and anti-alpha-Tubulin (TUBA) antibody (DM1A; Sigma). The band intensity of bovine A3Z3-HA (Figs 3 A,C, 4 B,D and 5 B,C and Supplementary Figure 5 ) was quantified using ImageJ software (http://rsbweb.nih.gov/ij/ ).
3
Quantifying Virus Infectivity and Protein Expression
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The culture supernatant harvested at 48 hours post-transfection was centrifuged to remove cells and produce virus suspensions. The infectivity of virus suspensions was measured by TZM-bl assay as previously described25 (link). Briefly, 100 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 (Applied Biosystems) and a 2030 ARVO X multilabel counter instrument (PerkinElmer) according to the manufacturers’ procedure. Western blotting was performed as previously described25 (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-alpha-Tubulin (TUBA) antibody (DM1A; Sigma).
4
Verifying LY6E-HA Expression via Western Blot
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To verify LY6E-HA expression in the transduced cells, Western blotting was performed as described previously [73 (link)]. Briefly, 1.0 × 106 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). Proteins in lysate were separated by SDS-PAGE with 14% acrylamide gel and then transferred to PVDF membrane. The following antibodies were used for protein detection: anti-HA antibody (3F10; Roche), anti-alpha-tubulin (TUBA) antibody (DM1A; Sigma). To verify the expression of endogenous LY6E in LY6E KO Jurkat-CCR5 cells, LY6E KO and control cells were treated with IFN-α (100 U/ml) for 24 h and then lysed in RIPA buffer. Lysates were processed and Western blotting for endogenous LY6E was performed as previously described with modifications as described below [41 (link)]. Briefly, proteins were separated on a low molecular weight tricine gel and transferred to PVDF membrane. LY6E was detected using a mouse anti-LY6E monoclonal antibody (4D8.6.7, Genentech), and beta-actin (ACTB) was detected using peroxidase-conjugated anti-ACTB monoclonal antibody (AC-15, Sigma).
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