SDS-electrophoresis and immunoblotting technique was used for CD9 and CD81 detection and was carried out using protocols based on standard methods [46 (link),47 (link)]. Samples containing protein equivalent to 5 × 106 sperm cells were run on a 4% stacking and 12% running SDS polyacrylamide gel using Precision Plus Protein™ Dual Color Standards (Bio-Rad, Hercules, CA, USA) as molecular weight markers. After transferring proteins onto a PVDF (Polyvinylidine Fluoride) membrane, nonspecific sites were blocked with SuperBlock (PBS) Blocking Buffer (Thermo Scientific). CD9 and CD81 molecules were identified by the primary polyclonal rabbit antibodies anti-CD9 (H-110) and anti-CD81 (H-121) diluted 1:500 in PBS, followed by a peroxidase goat anti-rabbit IgG secondary antibody (Bio-Rad) diluted 1:3000. Antibody reaction was visualised by chemiluminescent substrate Super Signal West Pico (Pierce, Rockford, IL, USA). As a negative control, blots were incubated with immunoglobulins from rabbit serum (Sigma-Aldrich) in the same concentration as primary antibody.
Superblock pbs blocking buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
SuperBlock (PBS) Blocking Buffer is a ready-to-use solution designed to block non-specific binding in Western blotting, ELISA, and other immunoassay techniques. The buffer contains a proprietary blend of proteins and surfactants that effectively minimize background signal and improve the specificity of antibody-based detection.
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Lab products found in correlation
Evos fl cell imaging system, by Thermo Fisher Scientific (2 mentions)
Nucblue fixed cell readyprobes reagent, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (2 mentions)
High binding 96 well plate, by Greiner (2 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (2 mentions)
Infinite m200, by Tecan (2 mentions)
Startingblock t20 tbs blocking buffer, by Thermo Fisher Scientific (2 mentions)
Sylgard 184 clear polydimethylsiloxane pdms, by Dow (2 mentions)
Fluorocarbon oil novec 7500, by 3M (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Precision plus protein dual color standard, by Bio-Rad (1 mentions)
405 ts microplate washer, by Agilent Technologies (1 mentions)
Goat anti human fc igg hrp, by Thermo Fisher Scientific (1 mentions)
Varioskan lux plate reader, by Thermo Fisher Scientific (1 mentions)
Primidone, by Merck Group (1 mentions)
Sodium valproate, by Merck Group (1 mentions)
Phenytoin sodium, by Merck Group (1 mentions)
Tween 20, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Pvdf membrane, by Cytiva (1 mentions)
28 protocols using superblock pbs blocking buffer
1
CD9 and CD81 Protein Detection in Sperm
2
SARS-CoV-2 Spike Protein ELISA Assay
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20μl of ectodomains (stabilized prefusion trimer) of SARS-CoV-2 or SARS-CoV, or the disulphide stabilized SARS-CoV-2 or SARS-CoV, were coated on 384 well ELISA plates at 1 ng/μl for 16 hours at 4°C. Plates were washed with a 405 TS Microplate Washer (BioTek Instruments) then blocked with 80 μl SuperBlock (PBS) Blocking Buffer (Thermo Scientific) for 1 hour at 37°C. Plates were then washed and 30 μl antibodies were added to the plates at concentrations between 4 × 10−8 and 10 ng/μl and incubated for 1 h at 37°C. Plates were washed and then incubated with 30 μl of 1/5000 diluted goat anti-human Fc IgG-HRP (invitrogen A18817). Plates were washed and then 30 μl Substrate TMB microwell peroxidase (Seracare 5120–0083) was added for 5 min at room temperature. The colorimetric reaction was stopped by addition of 30 μl of 1 N HCl. A450 was read on a Varioskan Lux plate reader (Thermo Scientific).
3
Quantitative CBZ Immunoassay Development
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CBZ, valproate sodium, phenytoin sodium, primidone, Tween-20 and bovine serum albumin (BSA) were purchased from Sigma-Aldrich (Shanghai, China). CBZ antibody clone CA1 was obtained from Bio-Rad Laboratories (Shanghai, China). CBZ bovine serum albumin conjugate was purchased from Unibiotest Co., Ltd. (Wuhan, China). ZebaTM spin desalting columns (7 K MWCO, 0.5 mL), 3,3′- diaminobenzidine (DAB) enhanced liquid substrate system tetrahydrochloride, and SuperBlock™ (PBS) blocking buffer were obtained from Thermo Scientific (Shanghai, China). The biotinylating kit was obtained from Genemore (Suzhou, China). The horseradish peroxidase (HRP) Conjugation Kit-Lightning-Link® (ab102890) was from Abcam (Shanghai, China). Single donor human serum off the clot and human whole blood was obtained from Innovative Research (Novi, MI, USA). The 96-well polystyrene black microplates were obtained from Greiner Bio-One GmbH (Shanghai, China). The Octet® K2 2-channel system and streptavidin sensors were purchased from Sartorius Stedim Biotech GmbH (Gottingen, Germany). Phosphate-buffered saline (PBS, 10 mM, pH 7.4), PBS buffer containing 0.02% (v/v) Tween-20 and 0.1% BSA (referred to as sample diluent (SD) buffer), and SD buffer containing 274 mM NaCl (referred to as high-salt SD buffer) were used in this study.
4
ASFV Protein Detection and Quantification
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HEK 293A cells were transfected as mentioned in Section Immunocytometry and cells were washed at 48 h post-transfection with 1x PBS and then lysed in RIPA buffer supplemented with protease inhibitor (Sigma, MO, USA). Following clarification by centrifugation, supernatants were prepared under reducing conditions in Laemmli buffer (Bio-Rad, CA, USA) containing 10% β-Mercaptoethanol followed by heat denaturation at 95°C for 5 min, fractionated by SDS-PAGE using a 7.5% Acrylamide/bis gel (ProtoGel, National Diagnostics, GA, USA), transferred to PVDF membranes (Amersham, MA, USA), and blocked for 1.5 h at room temperature in SuperBlock (PBS) Blocking Buffer (ThermoFisher, MA, USA). The membranes were incubated with a 1:10,000 dilution of ASFV-specific convalescent swine serum followed by exposure to a 1:8000 dilution of horseradish peroxidase-conjugated swine secondary antibody followed by chemiluminescence development (Pierce Chemiluminescent Plus Substrate Kit, Invitrogen, CA, USA) and detection using the Invitrogen iBright 1500 imaging system. Purified proteins TMSP7 and p62 that were previously validated served as the negative and positive controls, respectively.
5
Immunofluorescent Detection of Citrullinated Histone H3
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Human neutrophils cultured under the designated condition for 5 h were fixed with 4% paraformaldehyde (PFA) for 10 min and permeabilized with PBS containing 0.2% Tween20 for 10 min. After washing with PBS three times and incubating for 20 min in the SuperBlock (PBS) Blocking Buffer (ThermoFisher Scientific, MA, USA), cells were labeled with a rabbit anti-citrullinated histone H3 antibody (1:1000; ab5103, Abcam, MA, USA) for 1 h at room temperature. After washing with PBS containing 0.1% Tween20 three times, citrullinated histone H3 was labeled with Alexa Fluor 488 anti-rabbit IgG secondary antibody (1:1000; Invitrogen Inc., CA, USA) for 1 h at room temperature. After an additional 3 washes in PBS containing 0.1% Tween20, DNA was stained with DAPI (NucBlue Fixed Cell ReadyProbes Reagent, ThermoFisher Scientific, MA, USA). Slides were mounted in ProLong Gold anti-fade reagent (ThermoFisher Scientific, MA, USA). Images were acquired using the EVOS FL Cell Imaging System (Life technologies, CA, USA).
6
Immunohistochemical Detection of hnRNP G-T
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After deparaffinization and rehydration, endogenous peroxidase activity was blocked with 0.6% H2O2 in ethanol for 25 min. The slides were then treated by the antigen‐retrieval technique with autoclaving in 10 mM citrate buffer (pH 9.0) for 20 min at 121°C. After blocking any nonspecific reaction with SuperBlock™ (PBS) Blocking Buffer (Thermo Fisher Scientific), the sections were incubated with anti‐hnRNP G‐T antibody (sc‐101,134; SantaCruz) at 4°C overnight. This step was followed by sequential incubation with the secondary antibody (ImmPRESS UNIVERSAL Reagent, Anti‐Mouse/Rabbit Ig) (VECTOR LABORATORIES, INC.) for 1 h at room temperature and stained using a DAB Peroxidase Substrate Kit, ImmPACT (VECTOR LABORATORIES). Nuclei were stained with hematoxylin.
7
Microfluidic Cell Separation Protocols
8
Mannose-Purification of Surface Proteins
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The mannose-purification of clarified surface protein extracts of Ac and RAW 264.7 was scaled up to 96-well microplates previously sensitized with 50 µL of mannose solution (10 µg/mL) for 1 h at 37°C, and subsequently overnight at 4°C. After, the plates were blocked with blocking buffer (SuperBlock™ (PBS) Blocking Buffer, ThermoFisher), for 1 h at 37°C. Thereafter, plates were incubated with 200 µg/mL (50 µL/well) of phagocytes’ clarified extracts in DPBS, for 1 h at 37°C. Posteriorly, plates were washed three times with PBS to remove mannose unbound proteins. Then, mannose-attached proteins were recovered with stripping buffer (50 mM Tris-HCl, 50 mM DTT, and 2% SDS; pH 7.0) for 1 h at 70°C. Next, the content of each well was collected and dialyzed in dialysis cassettes (Slide-A-Lyzer™ 10K MWCO, ThermoFisher) against PBS/Ca+2/Mg+2 at 4°C, under 200 rpm shaking for three days, with daily buffer exchanges. Then, the mannose-purified surface proteins (MPPs) were collected, identified, and analyzed by Western blot. The protein banding pattern of the MPPs were compared between the distinct phagocytes.
9
CoVariant Protein Array Immunoassay
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The CoVariant protein arrays stored at −80 °C were warmed to room temperature and then washed with 1 × TBST (TBS buffer with 0.1% Tween 20) for 10 min. Arrays were blocked with SuperBlock (PBS) Blocking Buffer (Thermo Scientific, #37515) for 15 min and incubated with 50 μl of 50-fold diluted plasma in TBST with 1% bovine serum albumin for an hour. The CoVariant array was rinsed with TBST three times and incubated with a 50 μl cocktail of 30 ng/ml Cy3-labeled anti-human IgG+A + M antibodies (Jackson Laboratory #109-165-064), biotinylated human ACE2 39 ng/ml, and Cy5-conjugated streptavidin 1.8 ug/ml for an hour. After 3 times washing, the arrays were dried and scanned for Cy3 and Cy5 signals with power of 30% and 25% (Caduceus Biotechnology, #SpinScan). The fluorescence signals were aligned and exported as foreground minus background by GenePix Pro software.
10
Leptospira Protein Extraction and Analysis
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Cultures of Leptospira at mid to late-log phase of growth were centrifuged (10,000×g, 30 min, 4 °C) and the resulting pellet was washed twice with sterile PBS. Protein samples were processed for one-dimensional (1-D) SDS-PAGE on 12% acrylamide gels (BioRad) as per manufacturer’s guidelines. Proteins were visualized by staining with Sypro Ruby (Invitrogen, CA) as per manufacturer’s guidelines. For immunoblotting, proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes (BioRad), previously activated by methanol, by semidry transfer. Membranes were blocked with SuperBlock (PBS) Blocking Buffer (Thermo) for 1 h and then incubated with indicated primary antibody diluted in the blocking buffer (1:4000) for 1 h at room temperature. Membranes were then washed 3 times with PBS 0.1% Tween 20 (PBS-T) and incubated with horseradish peroxidase-conjugated secondary anti-rabbit IgG (1:4000) antibody in blocking buffer for 1 h at room temperature. After extensive washing, detection of antigen reactivity was revealed using Clarity and Clarity Max ECL (BioRad) and the luminescence generated was detected with a ChemiDoc MP Imaging System (BioRad).
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