20 µg of non-depleted pooled samples were loaded into pre-cast tris-gly gels AnyKD (Bio-Rad Laboratories Inc © , Hercules, CA, USA). After electrophoresis, the gels were incubated in transfer buffer (Bio-Rad Laboratories Inc © , Hercules, CA, USA) for 10 min. Transfer was performed in a Bio-Rad minitransfer system using a PVDF membrane previously activated with methanol for 1.5 h at 200 mA. After transfer, the gels were fixed in 40% ethanol / 10% acetic acid and stained. Membranes were blocked overnight with 5 % BSA (Roche Diagnostics © , S.L, Basilea, Switzerland) in TBS buffer (Bio-Rad Laboratories Inc © , Hercules, CA, USA) at 4º C. Membrane washing was performed with TBS buffer-Tween20 (5%). The primary antibodies were monoclonal antibodies anti-Apo A1 (Santa Cruz Biotechnology, Inc © , Dallas, TX, USA), antikininogen 1 (Abcam © , Cambridge, UK) and anti-vitronectin (Abcam © , Cambridge, UK) all diluted in 5% TBS-Tween20.
Tbs buffer
Manufactured by Bio-Rad
Sourced in United States
TBS buffer is a commonly used buffer solution in various laboratory applications. It is a Tris-buffered saline solution that provides a stable pH environment for various biomolecular interactions and assays. The core function of TBS buffer is to maintain a physiologically relevant pH and ionic strength for the preservation and analysis of biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Roche (1 mentions)
Transfer buffer, by Bio-Rad (1 mentions)
Anti apoa 1, by Santa Cruz Biotechnology (1 mentions)
Anti vitronectin, by Abcam (1 mentions)
Anykd, by Bio-Rad (1 mentions)
Sigmafast bcip nbt, by Merck Group (1 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Irdye 680rd goat anti rabbit igg, by LI COR (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Trans blot turbo rta midi 0.45 m lf pvdf transfer kit, by Bio-Rad (1 mentions)
2 mercaptoethanol, by Bio-Rad (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Intercept tbs blocking buffer, by LI COR (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
0.2 μm nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Mammalian protease inhibitor, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
4 protocols using tbs buffer
1
Immunoblot Analysis of Apolipoproteins
2
Colorimetric Detection of Alkaline Phosphatase
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Alkaline phosphatase activity detection was performed using the colorimetric assay SigmaFast™ BCIP®/NBT (Sigma, St. Louis, MO, USA) following manufacturer’s instruction. SigmaFast tablet was dissolved in 10 ml of deionized water, resulting in a ready-to-use solution. The iPSC culture medium was removed, and cells were washed with 1× TBS buffer (Bio-Rad, Richmond, CA, USA), and SigmaFast solution was added and incubated for 20 min at room temperature (RT). Finally, cells were washed with 1× PBS (Bio-Rad, Richmond, CA, USA), and positive colonies were observed and photo-documented with an inverted phase-contrast microscope (Olympus IX71).
3
Western Blot Analysis of Protein Lysates
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Cells were lysed in RIPA buffer (ThermoFisher, 89901) in the presence of Halt™ Protease and Phosphatase Inhibitor Cocktail (ThermoFisher, 78440), and protein concentration was measured using Pierce™ BCA Protein Assay Kit (ThermoFisher, 23227). Lysates were boiled for 5 min with 4x Laemmli Sample Buffer (Bio-Rad, 1610747) and 2-mercaptoethanol (Bio-Rad, 1610710). Lysates were resolved on SDS polyacrylamide gels and blotted onto PVDF membranes using Trans-Blot Turbo RTA Midi 0.45 µm LF PVDF Transfer Kit (Bio-Rad, 1704275). Transfer was run in Trans-Blot Turbo Transfer System (Bio-Rad, 1704150) using Mixed MW (1.3A-25V-7M) protocol. The membranes were blocked with Intercept™ (TBS) Blocking Buffer (LI-COR, 927-60001) for 1 h at room temperature and incubated with appropriate primary antibodies diluted 1:1000 in blocking buffer at 4°C overnight. After 3 washes with 0.05% tween20 (Bio-Rad) in TBS buffer (Bio-Rad), the membrane was incubated with IRDye® 800CW Goat anti-Mouse IgG (LI-COR, 926-32210) and IRDye® 680RD Goat anti-Rabbit IgG (LI-COR, 926-68071) secondary antibodies diluted 1:10000 in blocking buffer for 1 h at room temperature. Band images were visualized using ChemiDoc™ MP Imaging System (Bio-Rad, 12003154).
4
Quantification of Viral-Induced PD-L1 Expression
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A total of 5 × 106 cells cultured in six-well plates were infected with indicated VVs at MOI = 2. After incubation for 48 h, supernatants were harvested and clarified by centrifugation at 10,000 × g for 2 min. Cells were lysed in 1× RIPA buffer (Sigma-Aldrich, St Louis, MO) and 1× mammalian protease inhibitor (Sigma-Aldrich, St Louis, MO) for 15 min on ice and clarified by centrifugation at 10,000 × g for 2 min. Samples of both supernatants and cell lysates were mixed with 6× sodium dodecyl sulfate (SDS) sample buffer (Bioworld, Dublin, OH) and electrophoresed in a 4–20% gradient SDS–polyacrylamide gel (Thermo, Waltham, MA). The fractionated protein samples are transferred onto 0.2 μm nitrocellulose membrane (Thermo, Waltham, MA). The nitrocellulose membrane is blocked for 30–60 min at room temperature (RT) in TBS buffer (Bio-Rad, Irvine, CA) containing 5% nonfat milk. Immunodetection of iPDL1 is performed by incubation with RD800-conjugated goat anti-mouse IgG antibody (Licor, Lincoln, NE) at RT for 1 h, or with rat anti-mouse PD-1 (Biolegend, San Diego, CA) at 1 µg/mL for overnight at 4 °C followed by with an RD800-conjugated anti-Rat IgG (Licor, Lincoln, NE). The blots are detected with an Odyssey Imager (LI-CON, Lincoln, NE).
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