Cells were treated exactly as in the conditions used for imaging experiments before being lysed in non-denaturing lysis buffer (200 mM NaCl, 75 mM Tris-HCl, pH 7.4, 15 mM NaF, 1.5 mM Na3VO4, 7.5 mM EDTA, 7.5 mM EGTA, 1.5% (v/v) Triton X-100, 0.75% (v/v) NP-40, 50 μg/ml leupeptin, 50 μg/ml aprotinin, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride). Lysates were clarified by centrifugation at 10,000 g for 10 min at 4°C. Cell lysates were resolved under denaturing conditions by SDS-PAGE (4–12% Bis-Tris gels; Invitrogen) and transferred to nitrocellulose membrane. Membranes were blocked with 1x Blocking Buffer (Sigma) and incubated overnight at 4°C with the appropriate primary antibody in 5% BSA and then at room temperature for 1 h with the appropriate fluorophore-conjugated secondary antibody in 1x Blocking Buffer. Membranes were scanned using an infrared imaging system (Odyssey; LI-COR Biosciences). For quantification of western blots, mean intensity of each relevant band was measured using ImageJ. Loading was normalised to an appropriate loading control and backgrounds were subtracted. All conditions were normalised to the band with the highest intensity in each repeat, and then the mean normalised intensity was calculated across three independent repeats. Quantification of western blots was performed where explicitly stated.
Blocking buffer
Manufactured by Merck Group
Sourced in United States, Germany
Blocking buffer is a laboratory reagent used to prevent non-specific binding of antibodies or other proteins in various immunoassays, such as Western blotting, ELISA, and immunohistochemistry. It works by blocking unoccupied binding sites on the solid support, thereby reducing the likelihood of unwanted interactions and improving the specificity of the assay.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system, by LI COR (6 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Nitrocellulose membrane, by Cytiva (3 mentions)
Donkey irdye 800 conjugated anti mouse igg, by Rockland Immunochemicals (3 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (2 mentions)
Donkey alexa fluor 680 conjugated anti goat igg or anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Anti rhesus igg h l hrp secondary antibody, by Southern Biotech (2 mentions)
Benchmark plus microplate spectrophotometer, by Bio-Rad (2 mentions)
3 min protocol, by Bio-Rad (2 mentions)
Tgx gel, by Bio-Rad (2 mentions)
Image lab 3, by Bio-Rad (2 mentions)
Trans blot turbo system, by Bio-Rad (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Lsm800 confocal microscope, by Zeiss (2 mentions)
Odyssey, by LI COR (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Buffered formalin phosphate, by Thermo Fisher Scientific (2 mentions)
Slowfade antifade reagent, by Thermo Fisher Scientific (2 mentions)
46 protocols using blocking buffer
1
Western Blot Analysis of Protein Expression
2
Western Blot Protein Detection Protocol
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Protein samples resolved by SDS-PAGE were transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore) in ice-cold transfer buffer [25 mM Tris-HCl pH 8.3, 192 mM glycine, 1.3 mM SDS, 20% (v/v methanol] at 300 mA constant current for 2 hours. Membranes were blocked in 1× blocking buffer (Sigma) in TBS (10 mM Tris-HCl pH 8.0, 150 mM NaCl) and incubated overnight at 4°C with primary antibodies in 1× blocking buffer-TBS supplemented with 0.1% Tween20 (TBST). Membranes then underwent three 10-minute washes with TBST and were incubated for 1 hour at room temperature with secondary antibodies (anti-rabbit-IgG, anti-mouse-IgG or anti-chicken-IgY forms of IRDye 800 and/or IRDye 680; LI-COR Biosciences, Cambridge, UK) at a dilution of 1∶10,000 in TBST supplemented with 0.01% SDS. Membranes were given three 10-minute washes before being visualised and quantified using the Odyssey Infrared Imaging System (LI-COR Biosciences).
3
Immunofluorescent detection of SVDV in Sf9 cells
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The Sf9 cells were grown on glass slides and infected with the recombinant baculovirus at 0.1 MOI. At 24 h post-infection, infected cells were fixed with 10% buffered formalin phosphate (Fisher Scientific, USA) for 30 min at 37℃ and permeabilized with 20% acetone in PBS for 10 min at room temperature. The slides were blocked with 1× blocking buffer (Sigma-Aldrich) for 1 h at 37℃ and incubated with an in-house SVDV-specific Mab F44SVD [18 ] (1:100 in blocking buffer) for 1 h at room temperature. Slides were washed three times with PBS containing 0.05% Tween 20 and then incubated with Alexa Fluor 594 goat anti-mouse IgG (Invitrogen; 1:400 diluted in PBS) for 1 h at room temperature. After washing and air drying, slides were mounted with SlowFade antifade reagent (Invitrogen) and covered with coverslips. Microscopy was performed by using an Olympus FV1000 confocal microscope (Olympus, USA). Image acquisition and analysis were performed by using Fluo-View software (Olympus).
4
Western Blot Protein Analysis
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Cell lysates were diluted in 2× Laemmli sample buffer with β-mercaptoethanol and boiled at 95 °C for 5 min. Samples (20 μg protein) were loaded on TGX-gels (any kD; Bio-rad, Sundbyberg, Sweden) and electrophoresed with Tris/glycine/SDS buffer at 200 V. The molecular weight standard was a Precision Plus Protein Western C standard. After electrophoresis, the separated proteins were blotted to a polyvinyl difluoride (PVDF) membrane using the Trans-Blot Turbo system with pre-packed transfer packs and the 3-min protocol (Bio-rad). The blots were incubated in blocking buffer (Sigma Aldrich, Schnelldorf, Germany) at room temperature for 1 h. The primary antibody was diluted in blocking buffer (1 μg/mL) and the incubation lasted for 1 h or overnight at 4 °C. Then, the blots were washed in PBS-tween 20 (5 × 10 min, 25 rpm). The blots were incubated with secondary antibody (1 μg/mL) and a StrepTactin-HRP conjugate (2 μL) for 1 h. A strenuous washing protocol was used (5 × 10 min, 40 rpm). Finally, the blots were added a solution of luminol and peroxide buffer (Bio-rad) and the bands were detected by the ChemiDoc™ XRS+ system (Bio-rad) and analyzed with the software Image Lab™ 3.0.1 (Bio-rad).
5
Western Blot Protein Analysis Protocol
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Cells were lysed in non-denaturing lysis buffer (200 mM NaCl, 75 mM Tris-HCl, pH 7.4, 15 mM NaF, 1.5 mM Na3VO4, 7.5 mM EDTA, 7.5 mM EGTA, 1.5% (v/v) Triton X-100, 0.75% (v/v) NP-40, 50 μg/ml leupeptin, 50 μg/ml aprotinin, and 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride). Lysates were clarified by centrifugation at 10,000 g for 10 min at 4°C. Cell lysates were resolved under denaturing conditions by SDS-PAGE (4–12% Bis-Tris gels; Invitrogen) and transferred to nitrocellulose membrane. Membranes were blocked with 1x Blocking Buffer (Sigma) and incubated overnight at 4°C with the appropriate primary antibody in 5% BSA and then at room temperature for 1 h with the appropriate fluorophore-conjugated secondary antibody in 1x Blocking Buffer. Membranes were scanned using an infrared imaging system (Odyssey; LI-COR Biosciences). siRNA efficiency was quantified in ImageJ following appropriate normalization to a relevant loading control.
6
Immunofluorescent Staining of COVID-19 Liver Tissue
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Slides of liver tissue section from four nondiabetic COVID-19 fatal cases from HCFMRP-USP were fixed with 4% PFA, blocked with blocking buffer (Millipore, 20773-M), and stained with the primary antibodies (PA). The slides were washed with Tris-Buffered Saline with Tween-20 (TBS-T) and incubated with secondary antibodies (SA). Autofluorescence was quenched using Quenching Kit (Vector Laboratories™, SP-8400-15). Cells were fixed using 3.7% formaldehyde and blocked with 0.1% tween-20 plus 1% BSA in PBS. The cells were incubated with PA, washed with PBS, and incubated with SA. The slides were then mounted using the medium with DAPI (Vector Laboratories™, H-1200-10). Images were acquired by Axio Observer combined with LSM 780 confocal microscope (Carl Zeiss™) and analyzed with Image J. Antibodies were indicated in SI Appendix, Table S2 .
7
Evaluating Ras Signaling Modulation
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METH was provided by Ningbo Public Security Bureau. N-Acetyl-L-cysteine (NAC) was purchased from Sigma-Aldrich. All drugs were dissolved in saline (0.9% NaCl) and administered to cells directly or via intraperitoneal injection. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and DMSO were purchased from MedChemExpress. The BrdU cell proliferation kit was purchased from BioVision. The primary and secondary antibodies for Western blotting, including anti-Ras, anti-β-actin, anti-ERK, anti-pERK, anti-MEK, and anti-pMEK, were purchased from Proteintech. Polyvinylidene fluoride, blocking buffer, and ECL were purchased from Millipore. The Active Ras Pull-Down and Detection Kit was purchased from Thermo Fisher Scientific.
8
In Situ Proximity Ligation Assay
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In situ PLA was performed using a PLA kit (Sigma-Aldrich) according to the manufacturer’s instructions. In brief, cells were washed twice with ice-cold PBS and fixed in 3.65–3.8% formaldehyde for 30 min at room temperature. The cells were then permeabilized with 0.5% Triton X-100 for 10 min at room temperature. After washing three times with PBS, the cells were incubated in blocking buffer (Sigma-Aldrich) for 1 h at 37 °C. After blocking, the cells were incubated with the primary antibody diluted in antibody diluent (Sigma-Aldrich) for 1 h at 37 °C. The cells were then incubated with PLUS or MINUS PLA probes (Sigma-Aldrich) for 1 h at 37 °C. Ligation was performed for 30 min at 37 °C using a ligase specific for PLA (Sigma-Aldrich). After ligation, the cells were incubated in a polymerase solution containing a polymerase diluted in Duolink Amplification Green (Sigma-Aldrich) for 100 min at 37 °C. DAPI was used to stain the nuclei, and images were obtained using a Zeiss LSM 800 confocal microscope.
Where indicated, the PLA experiments were coupled to immunostaining experiments. After incubation of the cells in a polymerase solution during the PLA experiment, the cells were incubated with primary and secondary antibodies for immunostaining. The nuclei were stained with DAPI, and images were visualized using a Zeiss LSM 800 confocal microscope.
Where indicated, the PLA experiments were coupled to immunostaining experiments. After incubation of the cells in a polymerase solution during the PLA experiment, the cells were incubated with primary and secondary antibodies for immunostaining. The nuclei were stained with DAPI, and images were visualized using a Zeiss LSM 800 confocal microscope.
9
SDS-PAGE and Western Blot Analysis
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Protein samples were separated by SDS-PAGE (4–12% [wt/vol] NuPAGE Novex Bis-Tris gels; Invitrogen) at 200 V for 45 min. To visualize total protein, gels were incubated in Instant Blue (Expedeon) for 1 h and washed in water overnight at 4°C. For immunoblotting, gels were transferred onto nitrocellulose membrane (Whatman) and membranes were blocked with blocking buffer (Sigma-Aldrich) in PBS− for 1 h at RT. Membranes were incubated with appropriate concentrations of primary antibodies diluted in blocking buffer in TBS (10 mM Tris-HCl, pH 8.0, and 100 mM NaCl) supplemented with 0.05% (vol/vol) Tween-20 (TBS-T) overnight at 4°C. After three washes with TBS-T, membranes were incubated with appropriate secondary antibodies diluted in blocking buffer in TBS-T for 45 min at RT in the dark and were washed three times in TBS-T. Secondary antibodies used were donkey Alexa Fluor 680–conjugated anti–goat IgG, anti–mouse IgG, or anti–rabbit IgG (Life Technologies) and donkey IRDye 800–conjugated anti–mouse IgG (Rockland Immunochemicals). Membranes and stained gels were scanned using the Odyssey infrared imaging system (LI-COR), and total lane intensities were determined using Odyssey software (LI-COR).
10
Western Blot Protein Detection Protocol
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