HEK and Nomi EVs were boiled at 95 °C for 10 min and the concentration was determined using Pierce BCA Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL,USA). Then 25 μg of each sample was resolved by electrophoresis on 4–12% SDS-PAGE in Bis-Tris Plus (Thermo Scientific Pierce, Rockford, IL,USA) at 120 V. Next, the proteins were transferred to polyvinylidene fluoride membranes (PVDF) using the Invitrogen iBlot 2 Dry Blotting System. Membranes were blocked by incubation in 5% non-fat milk powder in 0.1% Tween 20 in Tris buffered saline (TBS-T) and incubated overnight at 4 °C with primary antibody against HSP-70 (1:500 Santa Cruz Biotechnology, D1519), Nanoluc (1:500, Promega, N700A), RFP (Thermo Fisher, MA5–15257) at 4 °C overnight. Then, the membranes were washed 3 times with TBS-T for 10 min and incubated with a secondary antibody (1:2500) at RT for 1 h. Membranes were then washed with 3 times TBS-T for 10 min. Bands were visualized by Pierce™ ECL Western Blotting Substrate (32106) in the Azure Biosystems C300 imaging equipment.
Bis tris plus
Manufactured by Thermo Fisher Scientific
Sourced in United States
Bis-Tris Plus is a buffer used in electrophoresis and other biochemical applications. It is a zwitterionic buffer with a pKa of 6.5 at 25°C, which makes it suitable for maintaining a neutral pH environment during experiments.
Automatically generated - may contain errors
Lab products found in correlation
Nanoluc, by Promega (2 mentions)
Iblot 2 dry blotting system, by Thermo Fisher Scientific (2 mentions)
Ecl western blotting substrate, by Azure Biosystems (2 mentions)
C300 imaging equipment, by Azure Biosystems (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Odyssey fc imager, by LI COR (2 mentions)
Odyssey imaging system, by LI COR (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Signalfire elite ecl, by Cell Signaling Technology (1 mentions)
Anti gapdh, by OriGene (1 mentions)
Horseradish peroxidase conjugated anti rabbit antibody, by GE Healthcare (1 mentions)
Anti β actin 8h10d10, by Cell Signaling Technology (1 mentions)
Anti rb clone g3 245, by BD (1 mentions)
Lumiglo peroxidase chemiluminescent substrate kit, by Cell Signaling Technology (1 mentions)
Image studio lite, by LI COR (1 mentions)
Anti sting, by Cell Signaling Technology (1 mentions)
Anti cgas, by Cell Signaling Technology (1 mentions)
Anti actin antibody, by Santa Cruz Biotechnology (1 mentions)
21 protocols using bis tris plus
1
Characterization of Extracellular Vesicles
2
Characterization of Extracellular Vesicles
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HEK and Nomi EVs were boiled at 95 °C for 10 min and the concentration was determined using Pierce BCA Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL,USA). Then 25 μg of each sample was resolved by electrophoresis on 4–12% SDS-PAGE in Bis-Tris Plus (Thermo Scientific Pierce, Rockford, IL,USA) at 120 V. Next, the proteins were transferred to polyvinylidene fluoride membranes (PVDF) using the Invitrogen iBlot 2 Dry Blotting System. Membranes were blocked by incubation in 5% non-fat milk powder in 0.1% Tween 20 in Tris buffered saline (TBS-T) and incubated overnight at 4 °C with primary antibody against HSP-70 (1:500 Santa Cruz Biotechnology, D1519), Nanoluc (1:500, Promega, N700A), RFP (Thermo Fisher, MA5–15257) at 4 °C overnight. Then, the membranes were washed 3 times with TBS-T for 10 min and incubated with a secondary antibody (1:2500) at RT for 1 h. Membranes were then washed with 3 times TBS-T for 10 min. Bands were visualized by Pierce™ ECL Western Blotting Substrate (32106) in the Azure Biosystems C300 imaging equipment.
3
Histone Modifications in BMDMs
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BMDMs were lysed with RIPA buffer and the whole-cell protein lysates were separated on pre-cast Bolt 12% Bis-Tris Plus gels (Invitrogen, NW00127BOX) and transferred to nitrocellulose membranes. Membranes were subsequently probed using the relevant primary (histone H3, H3K4me3, H3K27me3 and H3K9me2) and secondary antibodies (anti-IgG) and imaged using the Odyssey Fc Imager (Li-Cor). Images of blots were analysed with ImageJ to calculate the relative optical density (ROD = area/percentage). ROD was then standardized to the untrained/media control samples: adjusted ROD values.
4
Western Blot Analysis of Photosynthetic Proteins
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Thylakoids were loaded onto a pre‐cast gel [4–12% T (w/v) Bis‐Tris Plus, Invitrogen] based on their chlorophyll content. After electrophoresis, the proteins were transferred to a nitrocellulose membrane and stained with Ponceau S solution [0.1% (w/v) Ponceau S in 5% (v/v) acetic acid]. The stained membranes were imaged using a LAS 4000 Image Analyzer (ImageQuant Luminescent) under digital imaging (exposure time: 1/60 s). Afterward, the membranes were blocked with 10% (w/v) milk in TBST (20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween 20) for 1 hr and were incubated with different primary antibodies (all from Agrisera, Sweden) overnight in the dark at 4°C. The primary antibodies included: PsaA (AS06172), PSAG (AS204368), PSAK (AS04049), LHCA1 (AS01005), PsbA (AS10704), PsbC (AS111787), LHCB1 (AS01004), PSBS (AS09533), PetA (AS06119) and ATPC (AS08312). The membranes were washed with TBST for 4× 10 min and then incubated with the secondary antibody (goat anti‐rabbit IgG) for another hour. After washing with 4 × 5 min and 2 × 30 min to get rid of the unbound secondary antibody, the membranes were developed for chemiluminescence (SuperSignal West Pico, Thermo Scientific) using a LAS 4000 Image Analyzer. Densitometric analysis of the images was performed using the software ImageJ.
5
Western Blot Analysis of Microsomal Proteins
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Microsomal membrane fractions (50 μg/well) were loaded on a 4-12% Bis-Tris Plus or 10% Bis-Tris Plus gel (Invitrogen, USA) followed by gel electrophoresis at 130 V for 90 min. Resolved proteins were electroblotted onto a 0.45-μm protran nitrocellulose membrane (GE Healthcare, USA) at 12 mV for 60 min. Subsequently, the membrane was blocked in 5% non-fat milk with TBST (50 mM TRIS-HCl, 150 mM NaCl, 0.1% (v/v) Tween-20, pH 7.5) for 1 h. Membranes were incubated in anti-GAPDH (1:500 dilution, Acris, USA), anti-TRPM4 [38 (link)] (1:100, ab578), and anti-Calnexin (1:200, ab1039) antibodies at 4 °C for 24 h. Membranes were washed three times for 10 min in TBST and were then incubated in horseradish peroxidase-conjugated anti-rabbit antibody (1:50000, GE Healthcare, USA) at room temperature for 2 h. Membranes were washed two times for 10 min in TBST and once for 10 min in TBS (50 mM TRIS-HCl, 150 mM NaCl, pH 7.5). Afterwards, membranes were incubated in SignalFire Elite ECL (Cell Signaling, USA) for 1 min followed by the detection of chemiluminescence using digital imaging (GE Healthcare, ImageQuant LAS 4000 mini). Protein expression levels were measured by densitometry analysis using the ImageJ software.
6
Coomassie and Silver Staining of Membrane Proteins
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To charge the success of expression, purification, and reconstitution, LacY, YidC, SecYEG, and SecYEG-YidC samples were Coomassie Blue–stained (fig. S1) or silver-stained (fig. S2) after SDS-PAGE (4 to 12% Bis-Tris Plus gel; Invitrogen, Thermo Fisher Scientific, MA, USA).
7
Western Blot Analysis of E2F1 and Rb Proteins
8
Western Blot Analysis of Phospho-Akt
9
SDS-PAGE Immunoblotting of Viral Proteins
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Cells lysed in 1× RIPA buffer were resuspended in SDS sample buffer, separated by electrophoresis on Bolt 4%–12% Bis-Tris Plus gels (Invitrogen), blotted onto nitrocellulose membranes, and probed with the following antibodies: mouse monoclonal anti-p24 (CA) antibody (183-H12-5C; NIH AIDS reagents), mouse monoclonal anti-Actin antibody (Santa Cruz Biotechnology #sc-8432), the rabbit polyclonal anti-PQBP1 antibody (Bethyl Laboratories #A302-802A), or the following rabbit monoclonal antibodies: anti-cGAS (Cell Signaling Technology #15102S), anti-IFI16 (Cell Signaling Technology #14970S), anti-MAVS (Cell Signaling Technology #24930S), anti-MyD88 (Cell Signaling Technology #4283S), anti-STING (Cell Signaling Technology #13647S), anti-NONO (Millipore Sigma, #05–950) and anti-TREX1 (Cell Signaling Technology #15107S).
10
Western Blot Analysis of Protein Extracts
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Cultured explants were lysed on ice in RIPA (Radioimmunoprecipitation assay) buffer supplemented with halt protease (Thermofisher) and phosphatase cocktail inhibitors (P5726 and P0044, Sigma). Twenty µg of protein were loaded on an 8% Bis-Tris Plus precast polyacrylamide gel and run in 1X Bolt MES SDS running buffer (Invitrogen) using the Bolt System (Invitrogen Waltham, MA, USA). Transfers were performed using the iBlot 2 System (Invitrogen) and nitrocellulose gel transfer stacks. After the transfer, membranes were blocked in a 50:50 Odyssey Blocking (LI-COR, Lincoln, NE, USA): TBST (TBS and 0.1% Tween) solution at room temperature for at least 1 h and incubated with primary antibodies for 24 h at 4 °C with continuous agitation (Table 2 ). The following day, membranes were washed with TBST and incubated with fluorescent secondary antibodies diluted in blocking buffer for 1 h at room temperature. Final detection was obtained by enhanced fluorescence with a Chemidoc MP imaging system (Biorad, Hercules, CA, USA). Densitometry was analyzed using ImageLab software (Version 6.1, Biorad).
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