The cells were fixed in 4% (w/v) paraformaldehyde for 15–20 min at room temperature, followed by washing in PBS three times for 5 min each. After permeabilization with 0.2% (v/v) Triton X-100 for 15–20 min, the cells were again washed in PBS three times for 5 min each. Samples were incubated in StartingBlock (TBS) Blocking Buffer (Thermo Fisher Scientific) for 1 h at room temperature. Incubation with primary antibodies was performed overnight at 4°C. The following day, the cells were incubated with secondary antibodies for 1 h at room temperature. The cells were observed with a Zeiss LSM700 confocal microscope. Quantification of the immunofluorescence was performed with an In Cell Analyzer 6000 (GE Healthcare, Little Chalfont, UK).
Startingblock tbs blocking buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
StartingBlock (TBS) Blocking Buffer is a ready-to-use solution designed to block non-specific binding in immunoassays and western blotting procedures. It is a proprietary formulation that effectively reduces background signal and improves the signal-to-noise ratio in these applications.
Automatically generated - may contain errors
Lab products found in correlation
Complete protease inhibitor cocktail, by Roche (2 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
In cell analyzer 6000, by GE Healthcare (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Startingblock t20 tbs blocking buffer, by Thermo Fisher Scientific (1 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (1 mentions)
Image studio software, by LI COR (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Immobilon p transfer membrane, by Merck Group (1 mentions)
Sypro ruby, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase anti rabbit immunoglobulin g conjugate, by Merck Group (1 mentions)
Pro q emerald 300, by Thermo Fisher Scientific (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Free protease inhibitor cocktail tablet, by Roche (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Ripa lysis and extraction buffer, by Thermo Fisher Scientific (1 mentions)
11 protocols using startingblock tbs blocking buffer
1
Immunofluorescence Staining and Quantification
2
Western Blot Protein Extraction and Analysis
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Cell lysates were prepared in ice-cold lysis buffer [40mM HEPES pH=7.4, 1 mM ethylenediaminetetraaceticacid (EDTA), 120 mM sodium chloride, 50 mM bis-glycerophosphate, 1.0% Triton-X 100, 1.5 mM sodium orthovanadate, 50 mM sodium fluoride, and protease inhibitor cocktail (Sigma-Aldrich, P8340)]. Lysate samples were clarified by centrifugation for 10 min at 13,400 rpm and 4°C. Total protein concentration was determined using Protein Assay Dye Reagent Concentrate (Bio-Rad, 5000006). Proteins resolved by pre-cast 4–12% NuPage Bis-Tris Plus Midi gels (Invitrogen, WG1403BOX) or 3–8% NuPage Tris-Acetate Midi gels (Invitrogen, WG1603BOX) and electrotransferred onto either nitrocellulose or polyvinylidene difluoride membranes. Membranes blocked with StartingBlock (TBS) Blocking Buffer (Thermo, 37542) and primary antibodies diluted in StartingBlock T20 (TBS) Blocking Buffer (Thermo, 37543) at 4°C. After three 5 min 0.1% Tween20-TBS washes, membranes incubated in secondary antibodies StartingBlock T20 (TBS) Blocking Buffer for 1 hour at room temperature. Protein bands imaged using LI-COR Odyssey Infrared Imaging System and quantified with LI-COR Image Studio Software.
3
Leptospira Outer Membrane Enrichment and Characterization
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Fractionation of L. interrogans strain RJ19115 to enrich for outer membrane (OM) proteins was performed using Triton X-114 as previously described (Nally et al., 2001 (link)). OM enriched fractions were compared to whole leptospires by 1-D gel electrophoresis as previously described (Monahan et al., 2008 (link)). Proteins were visualized by staining with Sypro Ruby (Invitrogen, CA) and lipopolysaccharide was visualized by staining with Pro-Q Emerald 300 (Invitrogen, CA) as per manufacturer's guidelines. For immunoblotting, samples were transferred to Immobilon-P transfer membrane (Millipore, 220 Bedford, MA) and blocked overnight at 4°C with StartingBlock (TBS) blocking buffer (Thermo Scientific, CO). Membranes were individually incubated with indicated antisera (anti-LipL21, anti-LipL32 and anti-LipL41 at 1:4,000, anti-Treponema FlaA at 1:2,000, or a pool of sera from infected or non-infected rats at 1:1,000, in PBS-T for 1 h at room temperature), followed by incubation with horseradish-peroxidase anti-rabbit immunoglobulin G conjugate or horseradish-peroxidase anti-rat immunoglobulin G conjugate (Sigma, MO). Bound conjugates were detected using Clarity Western ECL substrate (BioRad, CA) and images acquired using a Bio-Rad ChemiDoc MP imaging system.
4
Quantitative Western Blot Analysis of Cadherin-13
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Frozen heart specimens were minced and resuspended in RIPA lysis and extraction buffer (Thermo Fisher, Tokyo, Japan) supplemented with complete EDTA-free protease inhibitor cocktail tablets (Roche, Basel, Switzerland). Lysates were homogenized with Tissue Lyser II (Qiagen) and sonicated to shear genomic DNA, and protein was then quantified by the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). Protein lysates were boiled with Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) at 95 °C for 5 min.
Protein samples were run on 8–16% MINI PROTEAN TGX gel (Bio-Rad), transferred to nitrocellulose membranes, and probed with the following polyclonal antibodies in Starting Block (TBS) Blocking Buffer (Thermo Fisher Scientific): anti-human cadherin-13 antibody (1:2000, R&D Systems, Minneapolis, MN, USA) and rat anti-beta-actin antibody (Abcam), followed by incubation with horseradish peroxidase-conjugated secondary antibodies at 1:5000 dilution. Signals were detected with an ECL system (Bio-Rad), visualized with ChemiDoc XRS and quantitated with ChemiDoc software (Bio-Rad).
Protein samples were run on 8–16% MINI PROTEAN TGX gel (Bio-Rad), transferred to nitrocellulose membranes, and probed with the following polyclonal antibodies in Starting Block (TBS) Blocking Buffer (Thermo Fisher Scientific): anti-human cadherin-13 antibody (1:2000, R&D Systems, Minneapolis, MN, USA) and rat anti-beta-actin antibody (Abcam), followed by incubation with horseradish peroxidase-conjugated secondary antibodies at 1:5000 dilution. Signals were detected with an ECL system (Bio-Rad), visualized with ChemiDoc XRS and quantitated with ChemiDoc software (Bio-Rad).
5
Western Blot Analysis of RUNX2 in Osteoblasts
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Proteins were extracted from lysates of MC3T3-E1 cells cultured in osteoblast differentiation medium for 12 days via the same method described above using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) with Halt Protease and Phosphatase Inhibitor Single-Use Cocktail (100×) (Thermo Fisher Scientific). The protein solution was centrifuged, and the supernatant was mixed with 4× Bolt LDS Sample Buffer (Thermo Fisher Scientific), separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) on a Bolt 4–12% Bis-Tris Plus gel (Thermo Fisher Scientific), and transferred to a polyvinylidene difluoride Membrane Filter Paper Sandwich (Thermo Fisher Scientific). The membranes were probed using an anti-RUNX2 antibody (Abcam, Cambridge, UK) and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology, Danvers, MA, USA) or an anti-β-actin HRP-conjugated antibody (Cell Signaling Technology), followed by incubation with StartingBlock (TBS) Blocking Buffer (Thermo Fisher Scientific). Subsequently, the membranes were incubated with ECL Select reagent (GE Healthcare, Little Chalfont, UK) and analyzed using an Image Quant LAS 4000 system (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
6
Staphylococcal Toxin Detection Protocol
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Supernatant dilutions consisted of 75% supernatant and 25% 4X Laemli’s reducing buffer (Boston BioProducts). Dilutions were denatured at 70°C for 10 minutes or at 100°C for 5 minutes. Gels were run for 35 minutes at 165V. Blots were transferred using a standard 7-minute procedure using iBlot 2 (Thermo Fisher Scientific).
Blots were blocked with StartingBlock (TBS) blocking buffer (Thermo Fisher Scientific) for 10 minutes and incubated in primary antibodies specific to different Staphylococcal toxins: Hla (Cat# Hla-ID-P), LukS-PV(Cat# LukS-ID-P), LukAB (cat# LukAB-ID-P), LukF-PV (Cat# LukF-IP-T01), TSST-1 (cat# TSST-1-ID-P-02), SEB (Cat-SEB-ID-P), and SEA (cat# SEA_ID-02) (all generated by IBT Bioservices) for 18 +/- 2 hours (26 (link)). This was followed by 2 rapid washes and a third 5-minute wash in 1X TBS-T (Thermo Fisher Scientific). Secondary antibody was diluted at 1:3000, and blots were incubated in it for 50-60 minutes. The blots were finally washed three times as described earlier. Blots incubated in AP- or HRP-conjugated secondary antibodies were washed in 1X TBS for 5 minutes. When using AP-conjugated secondary antibody (Bio-Rad), blots were developed using AP detection buffer (Bio-Rad) and imaged with a camera (Azure system). The blots incubated in HRP-conjugated secondary antibodies (KPL) were developed with ECL reagent (Azure) and imaged with Azure 600 imager.
Blots were blocked with StartingBlock (TBS) blocking buffer (Thermo Fisher Scientific) for 10 minutes and incubated in primary antibodies specific to different Staphylococcal toxins: Hla (Cat# Hla-ID-P), LukS-PV(Cat# LukS-ID-P), LukAB (cat# LukAB-ID-P), LukF-PV (Cat# LukF-IP-T01), TSST-1 (cat# TSST-1-ID-P-02), SEB (Cat-SEB-ID-P), and SEA (cat# SEA_ID-02) (all generated by IBT Bioservices) for 18 +/- 2 hours (26 (link)). This was followed by 2 rapid washes and a third 5-minute wash in 1X TBS-T (Thermo Fisher Scientific). Secondary antibody was diluted at 1:3000, and blots were incubated in it for 50-60 minutes. The blots were finally washed three times as described earlier. Blots incubated in AP- or HRP-conjugated secondary antibodies were washed in 1X TBS for 5 minutes. When using AP-conjugated secondary antibody (Bio-Rad), blots were developed using AP detection buffer (Bio-Rad) and imaged with a camera (Azure system). The blots incubated in HRP-conjugated secondary antibodies (KPL) were developed with ECL reagent (Azure) and imaged with Azure 600 imager.
7
Protein Expression and Immunoblotting
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To confirm TurboID bait expression and activity, 18 μg input or unbound fraction and 8 μl of the IP fraction of each sample was loaded on a 10% polyacrylamide gel. Following SDS-PAGE, proteins were transferred to 0.45-μm nitrocellulose membrane (GE Healthcare) for 2 h at 100V. Membranes were blocked using StartingBlock TBS blocking buffer (Thermo Fisher Scientific) mixed 1:1 with TBS-T (50 mM Tris–HCl pH 7.5, 150 mM NaCl supplemented with 0.1% Tween20) and subsequently incubated with antibodies against β-actin (1:20,000) and biotin (1:10,000). Immunoblots from nuclear fractionation experiments were incubated with antibodies against V5 (1:1000), GR (1:2000), lamin A/C (1:2000), and beta-actin (1:20,000).
Samples from siRNA treatments were lysed in ice-cold modified RIPA buffer (50 mM Tris pH 8.0, 200 mM NaCl, 0.5% NaDOC, 0.05% SDS, 1% NP-40, 2 mM EDTA) supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific 78440), briefly sonicated, and centrifuged at 20,000g for 10 min (4 °C) to remove cell debris. Protein concentrations were normalized using the Lowry-based DC Protein assay (Biorad 500112). Samples were loaded on 4 to 7.5% polyacrylamide gradient gels. Immunoblots were detected with antibodies against beta-actin (1:20,000), ZBTB20 (1:4000), CBP (1:1000), p300 (1:1000), NCOA1 (1:1000), NCOA2 (1:1000), and BRM (1:1000).
Samples from siRNA treatments were lysed in ice-cold modified RIPA buffer (50 mM Tris pH 8.0, 200 mM NaCl, 0.5% NaDOC, 0.05% SDS, 1% NP-40, 2 mM EDTA) supplemented with HALT protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific 78440), briefly sonicated, and centrifuged at 20,000g for 10 min (4 °C) to remove cell debris. Protein concentrations were normalized using the Lowry-based DC Protein assay (Biorad 500112). Samples were loaded on 4 to 7.5% polyacrylamide gradient gels. Immunoblots were detected with antibodies against beta-actin (1:20,000), ZBTB20 (1:4000), CBP (1:1000), p300 (1:1000), NCOA1 (1:1000), NCOA2 (1:1000), and BRM (1:1000).
8
Protein Immunoprecipitation from Transfected HEK293T Cells
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HEK293T cells, transiently transfected (calcium phosphate method) with the plasmids as indicated, were serum-starved overnight, followed by 3 h stimulation. Next, cells were lysed (50 mM Tris–HCl pH 7.5, 125 mM NaCl, 5% glycerol, 0.2% NP40, 1.5 mM MgCl2 and Complete Protease Inhibitor Cocktail (Roche)) and incubated overnight with anti-FLAG beads (anti-FLAG M2 affinity gel, Sigma Aldrich) on a rotor at 4 °C. These beads were blocked beforehand for 1 h at 4 °C, using undiluted StartingBlock™ (TBS) Blocking buffer (Thermo Scientific). After washing, the samples were eluted using Laemmli buffer, boiled for 5′ at 95 °C and stored at −20 °C. Finally, Western Blotting and antibody probing procedures were performed according to the guidelines of the company for the respective antibodies anti-HA (Roche), anti-FLAG (Sigma Aldrich), anti-E (Phadia) and anti-actin (as loading control) antibody (Sigma Aldrich). Imaging was done using KODAK films or the Amersham Imager 680 equipment.
9
Thrombus Protein Expression Analysis
10
SARS-CoV-2 rS Protein Antibody Inhibition Assay
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Human ACE2 receptor-blocking antibodies were determined by ELISA. Ninety-six well plates were coated with 1.0 μg mL−1 SARS-CoV-2 rS protein overnight at 4 °C. After washing with PBS-T and blocking with StartingBlock (TBS) blocking buffer (ThermoFisher Scientific), serially diluted serum from groups of immunized mice or baboons were added to coated wells and incubated for 1 h at room temperature. After washing, 30 ng mL−1 of histidine-tagged hACE2 (Sino Biologics, Beijing, CHN) was added to wells for 1 h at room temperature. After washing, HRP-conjugated anti-histidine IgG (Southern Biotech, Birmingham, AL, USA) was added, followed by washing and the addition of TMB substrate. Plates were read at OD 450 nm with a SpectraMax plus plate reader (Molecular Devices, Sunnyvale, CA, USA), and data were analyzed with SoftMax Pro 6.5.1 GxP software. Data shown in the graph are the average of triplicate wells. The % Inhibition for each dilution for each sample was calculated using the following equation in the SoftMax Pro program: 100 − [(MeanResults/ControlValue@PositiveControl)*100].
Serum dilution versus % Inhibition plot was generated and curve fitting was performed by four parameter logistic (4PL) curve fitting to data. Serum antibody titer at 50% inhibition (IC50) of hACE2 to SARS-CoV-2 rS protein (rS-WU1 or rS-Beta) was determined in the SoftMax Pro program.
Serum dilution versus % Inhibition plot was generated and curve fitting was performed by four parameter logistic (4PL) curve fitting to data. Serum antibody titer at 50% inhibition (IC50) of hACE2 to SARS-CoV-2 rS protein (rS-WU1 or rS-Beta) was determined in the SoftMax Pro program.
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