Crude GBS whole cell (WC) extracts, purified rSip or nature Sip were subjected to 12% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots. For WC samples, a 10 ml overnight culture was centrifuged to obtain the pellet, which was added with 25 μl of sample buffer (0.2 M Tris-HCl (pH 6.8), 1% SDS, 2% mercaptoethanol, 10% glycerol, 0.001% bromophenol blue) and then boiled for 5 min, and after centrifugation, 10 μl of supernatant was applied to the gel. After electrophoresis, protein bands were visualized by staining with coomassie brilliant blue (CBB) or transferred to nitrocellulose membranes for western blotting method using anti-rSip McAbs. For western blotting, the membrane was treated as follows: blocked with phosphate-buffered saline (PBS, pH 7.4) containing 3.0% BSA (Sigma-Aldrich, Beijing, China) and 0.1% Tween 20 (Sigma-Aldrich, Beijing, China) for 1 h at 24 °C, washed 3 times with PBS containing 0.1% Tween 20, incubated with 100 ng/ml of anti-rSip antibodies at 4 °C for 1 h, washed 3 times with PBS containing 0.1% Tween 20, incubated with 2,000-fold-diluted horseradish peroxidase (HRP)-conjugated anti-mouse IgG (Sigma-Aldrich, Beijing, China) at 24 °C for 1 h, washed 3 times with PBS containing 0.1% Tween 20, and then soaked in the TMB membrane peroxidase substrate system (Sigma-Aldrich, Beijing, China) for color development.
Tween 20
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Tween 20 is a non-ionic detergent commonly used in biochemical applications. It is a polyoxyethylene sorbitan monolaurate, a surfactant that can be used to solubilize and stabilize proteins and other biomolecules. Tween 20 is widely used in various laboratory techniques, such as Western blotting, ELISA, and immunoprecipitation, to prevent non-specific binding and improve the efficiency of these assays.
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Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (476 mentions)
Triton x 100, by Merck Group (287 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (143 mentions)
Sodium chloride (nacl), by Merck Group (105 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (101 mentions)
Tween 80, by Merck Group (96 mentions)
Phosphate buffered saline (pbs), by Merck Group (89 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (85 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (84 mentions)
Glycerol, by Merck Group (72 mentions)
Methanol, by Merck Group (72 mentions)
Paraformaldehyde (pfa), by Merck Group (63 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (59 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (59 mentions)
Ethanol, by Merck Group (58 mentions)
Sodium hydroxide (naoh), by Merck Group (55 mentions)
Glycine, by Merck Group (53 mentions)
Sucrose, by Merck Group (53 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (51 mentions)
Nitrocellulose membrane, by Bio-Rad (49 mentions)
3 102 protocols using tween 20
1
SDS-PAGE and Immunoblot Analysis of Sip Protein
2
Bikinin Modulates Barley Growth
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Barley at the stage described above (1st and 2nd leaf of approximately equal length) was treated with 10 µM (BK10), 50 µM (BK50), or 100 µM bikinin (BK100) or 0.11% DMSO as a control solvent solution (CK). All solutions were prepared as follows:
BK10: 0.98 µL 91.5 mM bikinin, 8.85 µL 100% DMSO, 25 µL Tween 20 (Sigma-Aldrich, Schnelldorf, Germany), 9 mL deionized water;
BK50: 4.92 µL 91.5 mM bikinin, 4.91 µL 100% DMSO, 25 µL Tween 20, 9 mL deionized water;
BK100: 9.83 µL 91.5 mM bikinin, 25 µL Tween 20, 9 mL deionized water; or
CK (0.11% DMSO): 9.83 µL 100% DMSO, 25 µL Tween 20, 9 mL deionized water.
3
Choroid Plexus Immunostaining Protocol
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The choroid plexus was explanted 48 h post adoptive cell transfer from the fourth ventricle and transferred on glass object slides and incubated in PBS (PAN Biotech) + tween20 (0.3%; Sigma) at RT for 5 min, washed twice in PBS (PAN Biotech) for 5 min and fixed in PBS (PAN Biotech) + PFA (2,2%; Sigma), glucose (2%, Sigma), sodium acide (0.02%; Sigma) for 20 min at RT. Choroid plexus were then rinsed in PBS (PAN Biotech), fixed in methanol (100%, Sigma) for 6 min at RT, washed twice in PBS for 5 min and blocked in PBS (PAN Biotech) + BSA (1%; PAN Biotech), tween20 (0.3%; Sigma), normal goat serum (10%; Sigma) for 30 min at RT. The staining was performed in PBS (PAN Biotech) + tween20 (0.3%; Sigma) + primary antibody for 2h at RT (rat anti CD31; 1:100; BD). After washing the tissue twice in PBS (PAN Biotech) for 5 min, secondary stainings were performed using anti rat secondary antibodies (donkey anti rat-Alexa647; life technologies). Finally, the tissue was stained with DAPI (1 µg/ml; Sigma) and embedded in fluorescent mounting media (Dako).
4
Quantifying Astrocyte Cytoskeleton and Protein Levels
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Day 67 GLAST-positive astrocytes were thawed and seeded into an hESC-qualified matrigel coated 96-well plate at 4e4 cells/cm2 for 3 technical replicates per biological (differentiation) replicate per cell line. Two days post seeding, cells were fixed using 4% paraformaldehyde (Electron Microscopy Sciences) in PBS for 20 minutes at room temperature. After washing cells three times with 0.1% Tween 20 (Sigma-Aldrich) in PBS, cells were permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) in PBS for 30 minutes at room temperature. Cells were then blocked for 1 hour in 5% goat serum (Gibco), 3% BSA (Gibco) in PBS for 1 hour at room temperature. Cells were incubated with Phalloidin-iFluor790 (Abcam, ab176763) at 1:1000 for 1 hour at room temperature, followed by washing three times with 0.1% Tween 20 (Sigma-Aldrich) in PBS. CellTag700 (LI-COR, 926–41090) at 1:500 was added as a total protein stain (for normalization purposes) and incubated for 1 hour at room temperature. Cells were then washed three times with 0.1% Tween 20 (Sigma-Aldrich) in PBS, then solution was completely removed from wells. Wells were immediately imaged using the LI-COR Odyssey CLx and analyzed using LI-COR’s Empiria Studio Software.
5
Immunofluorescence Staining Protocol
6
Immunofluorescence Labeling Protocol
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Samples were rinsed with PBS and fixed for 30 min with 4% prewarmed paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in PBS. Cells were permeabilised for 5 min with 0.2 % (Table 1, protocol A) or 0.5 % (protocol B) Triton X-100 (Sigma-Aldrich) in PBS. Blocking was performed either in 1% bovine serum albumin (BSA; w/v; Sigma-Aldrich), 0.5 % Tween-20 (Sigma-Aldrich) in PBS for 1 h at 37 • C (protocol A) or in 5% BSA (Sigma-Aldrich) for 30 min at RT(protocol B), depending on the antibody used (Table 1). Primary antibodies were applied overnight at 4 • C in 0.5 % BSA/0.5 % Tween-20 in PBS (protocol A) or 5% normal goat serum (v/v, Sigma-Aldrich)/0.5 % Tween-20 (Sigma-Aldrich) in PBS (protocol B). After washing away the primary antibodies, secondary antibodies were applied for 1 h in the same antibody incubation mixture. DAPI (Sigma-Aldrich) was used to stain nuclei of the cells. Imaging was performed on a Leica DMi8 microscope system (Leica, Wetzlar, Germany) using the appropriate Leica Software (LAS X). Images were further processed in ImageJ (version 1.51n; Rasband, 1997 Rasband, -2018 [25] [25] ).
7
Western Blot Analysis of Protein Samples
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Proteins were separated on polyacrylamide gel and transferred to nitrocellulose membrane (ThermoFisher Scientific). Membranes were blocked with 5% bovine serum albumin in PBS, with 0.1% Tween-20 (Sigma-Aldrich) for 40 min at RT, incubated overnight at 4 °C with the primary antibodies (dilution 1/1000e), washed in PBS-0.1% Tween-20, incubated further with HRP-conjugated secondary antibody (400 ng/mL) for 1 h at RT and washed again before analysis using Immobilon Western Chemiluminescent HPR Substrate system (Millipore, Molsheim, France). The chemiluminescent emission was registered by imageQuant LAS 4000 camera (GE Healthcare Life Science, Vélizy, France). Uncropped scans of key immunoblots are shown in Supplementary Fig. 7 .
8
SARS-CoV-2 nsp12 GTase Activity Assay
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SARS-CoV-2 nsp12 GTase activity assays were set up as described above, except that 1 mM GTP was used instead of α-32P-GTP, the diphosphorylated RNA concentration was increased to 10 μM and the reaction volume was increased to 5 μl. Reactions were incubated at 30°C for 240 min then heated to 95°C for 3 min. 500 nM SARS-CoV-2 nsp14 and 0.1 mM S-adenosylmethionine (NEB) were then added and reactions were incubated at 30°C for a further 60 min, then heated to 95°C for 3 min.
Reaction mixtures were dotted onto nylon membrane and allowed to air dry for 10 min, then heated to 80°C for 10 min under a light vacuum. Membranes were blocked in 1× PBS, 0.1% Tween-20 (Sigma) and 2.5% skimmed milk powder (Marvel), then incubated with mouse anti-m7G primary antibody (MBL; cat. RN016M) followed by goat anti-mouse HRP-conjugated secondary antibody (Sigma; cat. A4416) in blocking buffer. Membranes were washed 3× in 1× PBS, 0.1% Tween-20 then HRP activity was detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and exposure on Super RC Fuji Medical X-ray film (Kodak). Data were analysed using ImageJ and Prism 9 (GraphPad).
Reaction mixtures were dotted onto nylon membrane and allowed to air dry for 10 min, then heated to 80°C for 10 min under a light vacuum. Membranes were blocked in 1× PBS, 0.1% Tween-20 (Sigma) and 2.5% skimmed milk powder (Marvel), then incubated with mouse anti-m7G primary antibody (MBL; cat. RN016M) followed by goat anti-mouse HRP-conjugated secondary antibody (Sigma; cat. A4416) in blocking buffer. Membranes were washed 3× in 1× PBS, 0.1% Tween-20 then HRP activity was detected using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and exposure on Super RC Fuji Medical X-ray film (Kodak). Data were analysed using ImageJ and Prism 9 (GraphPad).
9
Functionalized Magnetic Nanoparticles for Biosensing
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We purchased FeCl3.6H2O and sucrose from Sangon Biotech Co., Ltd. (China). CH3COONa.3H2O, ammonia, ethylene glycol, tetraethylorthosilane (TEOS), and 3-aminopropyl) triethoxysilane (APTES) were provided by Aladdin Industrial Co., Ltd. (China). 4-(N-Maleimidomethyl) cyclohexane-1-carboxylic acid 3-sulfo-N-hydroxysuccinimide ester sodium salt (sulfo-SMCC), 1-(3-(dimethylamino)-propyl)-3-ethylcarbodiimide hydrochloride (EDC), N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), invertase (β-fructose-dase, from Baker’s yeast), Tris (2-car-boxyethyl) phosphine hydrochloride (TCEP), and Tween-20 were obtained from Sigma-Aldrich Co., Ltd. (USA). Reagents used in the polyacrylamide gel electrophoresis experiment and DNA sequences were provided by Sangon Biotechnology Co., Ltd. (Shanghai, China). The laboratory water was deionized water (≥ 18 MΩ·cm, Millipore).
Other buffer solutions were also employed:
Other buffer solutions were also employed:
Buffer A: 0.1 M sodium phosphate buffer including 0.1 M NaCl (pH 7.4)
Buffer B: 0.1 M sodium phosphate buffer including 0.1 M NaCl and 0.05% Tween-20 (pH 7.4)
Buffer C: 0.05 M HEPES including 1.5 M NaCl (pH 7.0)
Cu-Enz (5′-GGTAAGCCTGGGCCTCTTTCTTTTTAAGAAAGAAC-3′)
Cu-Sub (5′-biotin-(A)9AGCTTCTTTCTAATACGGCTTACC-(A)9(CH2)6SH-3′
10
Immunoblot Analysis of Sorted Monocytes
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Sorted monocytes were lysed in 2× Laemmli with 0.1 M dithiothreitol, boiled 10 min at 95 °C, and sonicated to collect total proteins, which were separated on polyacrylamide gel and transferred to nitrocellulose membrane (Thermo Fisher Scientific). Membranes were blocked with 5% bovine serum albumin in PBS, with 0.1% Tween-20 (Sigma-Aldrich) for 40 min at room temperature, incubated overnight at 4 °C with the primary antibodies, washed in PBS-0.1% Tween-20, incubated further with horse radish peroxidase-conjugated secondary antibody (400 ng/ml) for 1 h at room temperature and washed again before analysis using Immobilon Western Chemiluminescent HPR Substrate system (Millipore, Molsheim, France). The chemiluminescent emission was registered by imageQuant LAS 4000 camera (GE Healthcare Life Science, Vélizy, France). Uncropped scans of key immunoblots are shown in Supplementary Figure 11 .
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