Vero cells were lysed in Pierce RIPA buffer (Thermo Scientific, Waltham, MA, USA) and 20 μg of protein was separated using Bolt 4–12% Bis–Tris Plus (Invitrogen). Proteins were detected by probing the membranes with 1:1000 anti-SARS-CoV-2 nucleocapsid (Sino Bio., Beijing, China) and 1:2000 anti-GAPDH (Cell Signaling Technologies, Danvers, MA, USA) antibodies. Protein transfer was performed using iBlot 2 (Invitrogen) and iBlot 2 PVDF Regular Stacks (Invitrogen). Membranes were incubated with 1:2000 goat anti-rabbit antibody (Cell Signaling) conjugated with horseradish peroxidase for 1 h. Then, membranes were washed five times with tris-buffered saline with 5% Tween 20. Thereafter, the blots were detected using Super Signal (Thermo Scientific).
Bolt 4 12 bis tris plus
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Bolt 4–12% Bis-Tris Plus is a pre-cast polyacrylamide gel used for protein electrophoresis. It is designed for the separation and resolution of proteins in the molecular weight range of 10 to 300 kDa.
Automatically generated - may contain errors
Lab products found in correlation
Seeblue plus2 pre stained protein standard, by Thermo Fisher Scientific (3 mentions)
Ecl detection reagent, by GE Healthcare (2 mentions)
Nupage lds sample buffer, by Thermo Fisher Scientific (2 mentions)
Easy bradford, by Bio-Rad (2 mentions)
Wga glycoprotein isolation kits, by Thermo Fisher Scientific (2 mentions)
Iblot2, by Thermo Fisher Scientific (1 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions)
Iblot 2 pvdf regular stacks, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Chemidoc mp system, by Bio-Rad (1 mentions)
Anti β actin, by MP Biomedicals (1 mentions)
Anti snail1, by Santa Cruz Biotechnology (1 mentions)
Anti zeb1, by Abcam (1 mentions)
Anti p yap, by Cell Signaling Technology (1 mentions)
Anti rac1, by Cell Biolabs (1 mentions)
Anti rhoa, by Cell Biolabs (1 mentions)
Anti cdc42, by Cell Biolabs (1 mentions)
Anti yap, by Santa Cruz Biotechnology (1 mentions)
17 protocols using bolt 4 12 bis tris plus
1
Western Blot Analysis of SARS-CoV-2 Nucleocapsid
2
Western Blot Analysis of Cell Signaling
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The whole-cell extract was obtained in RIPA buffer in the presence of standard protease and phosphatase inhibitors. The protein content was determined with a protein assay reagent (Bio-Rad Laboratories Inc., Hercules, CA, USA), using bovine serum albumin as a standard. Equal protein content of total cell lysates was resolved on polyacrylamide gel (Bolt 4–12% Bis-Tris Plus Invitrogen, Carlsbad, CA, USA), electro-transferred to PVDF membranes (iBlot Invitrogen, Carlsbad, CA, USA) and incubated with specific primary antibodies: anti-YAP (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; sc #101199; dil.1:1000); anti-p-YAP (Cell Signaling Technology, Inc., Beverly, MA, USA; #13008; dil.1:1000); anti-β Actin (MP Biomedicals, Santa Ana, CA, USA; # 69100; dil.1:10000); anti-Rac1, anti-RhoA and anti-Cdc42 (all Cell Biolabs, San Diego, CA, USA; STA-405; dil.1:500); anti-Zeb1 (Abcam, Cambridge, MA, USA; #180905; dil.1:2000); anti-Snail1 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; sc-271977; dil.1:1000); anti-GSN (Sigma-Aldrich, St Louis, MO, USA; G-4896, dil.1:1000). Membranes were developed using ECL detection reagents (GE Healthcare Life Sciences, Uppsala, Sweden) on a UVITEC imaging system (UVITEC, Cambridge, UK) or ChemiDocMP system (Bio-Rad Laboratories Inc.). Quantitative analysis of western blots was performed using ImageJ (NIH) software [23 (link)].
3
Western Blot Analysis of Adipose Tissues
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Brain and brown adipose tissues were homogenized in RIPA buffer (Thermo Fisher Scientific) with phosphatase and protease inhibitor cocktails (Pierce Protease and Phosphatase Inhibitor Mini Tablets, EDTA-Free) added with 1% Triton (Euromedex, life sciences products) for the brown adipose tissues. Proteins were run on a polyacrylamide gel (Bolt 4–12% Bis Tris plus, Invitrogen by Thermo Fisher Scientific) and transferred to a nitrocellulose membrane (GE Healthcare Life Science). Primary antibodies were incubated overnight at 4 °C and were as follows: UCP1 (1:1000, Cell Signalling technology, #14670); p44/42 MAPK (1:1000, Cell Signalling technology, #9102); phospho-p44/42 MAPK (1:1000, Cell Signalling technology, #9101); GAPDH (1:1000, Invitrogen#PA1987). Signals were detected using Super Signal West Pico (Thermo Fisher Scientific, #34080) and bands were analyzed with ImageJ.
4
Evaluating Recombinant SARS-CoV-2 Protein Purity
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Expression and purity of recombinant S1 and RBD fusion proteins were evaluated by western blot and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified samples were added to sample loading dye NuPAGE LDS sample buffer and reducing agent (both Invitrogen) and heated to 70°C for 10 min. Samples were loaded into pre-cast polyacrylamide gels (Bolt 4–12% Bis-Tris Plus; Invitrogen) and run at 200 V for 40 min. Visualization of protein samples on acrylamide gels was performed using Coomassie Brilliant Blue G250 stain (Merck). Gels were stained overnight with agitation, and destaining solution (30% methanol and 10% acetic acid) was added for 1 h at room temperature. After separation by SDS-PAGE, proteins were transferred to a nitrocellulose membrane using a dry transblotter (Invitrogen). The membrane was blocked for 30 min (PBS containing 5% fat free milk and 0.1% Tween 20) at room temperature, followed by incubation with mouse anti-rabbit horseradish peroxidase (1:5,000; Sigma) for 1 h at 37°C with agitation. The membrane was washed four times using wash buffer (PBS with 0.1% Tween 20) at room temperature for 15 min, and developed using TMB solution (1-step ultra TMB-blotting solution, Thermo Scientific) in the dark for 30 min.
5
Glycoprotein Isolation and Purification
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The resuspended Euglena cells from the culturing (1 ml) were diluted with 5x binding/wash buffer (0.25 ml) containing phenylmethylsulfonyl fluoride (2 mM) and lysed by sonication (3 × 10 s, 25% amplitude, 30 s off between each pulse) and centrifuged (5 min, 1000 × g). Not all cells were lysed. Total lysate containing the equivalent of 1.1 mg of protein (Easy Bradford BioRad, BSA standards) was then used for glycoprotein purification using both ConA and wheat germ agglutinin (WGA) Glycoprotein Isolation Kits (Thermo Scientific) according to the manufacturer’s instructions. Protein quality was assessed using silver-stained SDS-PAGE (Bolt 4%–12% Bis-TRIS plus, Invitrogen) using SeeBlue Plus2 Prestained Protein Standard (Thermo Fisher Scientific) as the standard.
6
Western Blot Analysis of HCoEpC Cells
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Mock- and EBV-infected HCoEpC were washed twice in PBS, pelleted and lysed in RIPA buffer (NaCl 150 mM, NP40 1%, Tris–HCl pH8 50 mM, deoxycholic acid 0.5%, SDS 0.1%) containing protease and phosphatase inhibitors. 10 µg of proteins or 30 µg for EBNA1 detection were loaded on a precast polyacrylamide gel (Bolt™ 4–12% Bis-Tris Plus, Invitrogen, Waltham, MA, USA) and then transferred to a nitrocellulose membrane (Amersham ™), that was subsequently incubated for 30 min in a blocking solution (PBS, 0.1% Tween 20, 2% BSA). Next, membranes were incubated with primary antibody for 1 h at room temperature or overnight at 4 °C. After three washing in PBS-0.1% Tween 20 (washing solution), membranes were incubated for 30 min with a secondary antibody conjugated to horseradish peroxidase. Finally, membranes were washed three times with washing solution and protein detection as well as densitometric analysis were performed through a chemiluminescence kit Western Bright ECL (Advansta, Menio Park, CA, USA) and using ImageJ software, respectively.
7
Western Blot Validation of Proteomic Data
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Proteomic datasets from computational data analysis were validated by western blot assay. In brief, 20 μg of protein with sample buffer was loaded onto a Bolt 4 ~ 12% Bis-Tris Plus (Invitrogen, Carlsbad, CA, USA) gel and separated at 165 V for 35 min. Then proteins were transferred from the gel to 0.45 μm nitrocellulose membrane (BioRad, Hercules, CA, USA) using the Xcell-II blot module (Invitrogen, Carlsbad, CA, USA) at 25 V for 2 hr. The membrane was blocked for one hour at room temperature with blocking buffer, 5% dried non-fat milk (BioRad, Hercules, CA. USA) dissolved in Tris-buffered saline containing 0.1% Tween-20 (TBST). Membranes were incubated with primary antibody at 1:1,000 ~ 1:5,000 dilutions in blocking buffer overnight at 4°C. Blots were washed with TBST for 15 min 3 times. Membranes were incubated in the appropriate secondary antibody conjugated to HRP for 1 hr at room temperature. Blots were washed with TBST 3 times for 15 min. Chemiluminescent detection was accomplished using Amersham ECL prime western blotting detection reagent (GE Healthcare, Fairfield, CT, USA) and the UVP Biospectrum 500 Imaging System (Upland, CA, USA).
8
Protein Extraction and Western Blot
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Total protein extracts were isolated with NP-40 buffer (1% (v/v) NP-40, 0.15 M NaCl, 10 mM EDTA, 10 mM NaN3, 10 mM Tris-HCl pH 8) containing cOmplete™ Protease Inhibitor Cocktail (Roche, Basel, Switzerland, Cat#116974980001). The protein extracts (20 μg) were separated on SDS PAGE (Bolt™ 4–12% Bis-Tris Plus; Invitrogen; Cat#NW04120BOX) and transferred to Immobilon™-P PVDF membrane (Merck Millipore; Darmstadt, Germany, Cat#IPVH00010). Following the blocking in 5% (w/v) bovine serum albumin, the membranes were probed with primary antibodies FLAG-HRP (1:1000; Merck; Cat#F1804) or GAPDH mouse monoclonal antibody (1:5000; Abcam; Cambridge, UK, Cat#ab8245) and secondary antibody (1:5000; Merck Millipore; Cat#AP192P). To remove N-linked glycosylation moieties from cell lysates, equal quantities of protein were incubated with PNGase F (New England Biolabs; Ipswich, MA, USA, Cat#P0704S) at 37 °C for 24 h, prior to running on SDS-PAGE gels.
9
Virus Purification and Western Blot
10
Euglena Glycoprotein Purification Protocol
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The resuspended Euglena cells from the culturing (1 ml) were diluted with 5x Binding/Wash buffer (0.25 ml) containing phenylmethylsulfonyl fluoride (2 mM) and lysed by sonication (3 x 10 s, 25% amplitude, 30 s off between each pulse) and centrifuged (5 min, 1000 xg). Not all cells were lysed. Total lysate containing the equivalent of 1.1 mg of protein (Easy Bradford BioRad, BSA standards) was then used for glycoprotein purification using both ConA and WGA Glycoprotein Isolation Kits (Thermo Scientific) according to the manufacturer's instructions. Protein quality was assed using silver stained SDS-PAGE (Bolt 4-12% Bis-TRIS plus, Invitrogen) using SeeBlue Plus2 Pre-stained Protein Standard (Thermo Fisher Scientific) as the standard.
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