A 3 ml volume of pseudoparticles was pelleted using a TLA-55 rotor with an Optima-MAX-E ultracentrifuge (Beckman Coulter) for 2 hours at 42,000 rpm at 4°C. untreated particles were resuspended in 30 μl DPBS buffer. Pseudopartices were generated as described in the pseudoparticle generation section, with the furin inhibitor dec-RVKR-CMK (Cat:35–011, Tocris) being added during transfection to select wells. For the + furin treated MLVpps, particles were resuspended in 30 μL of furin buffer consistent in 20 mM HEPES, 0.2 mM CaCl2, and 0.2 mM β-mercaptoethanol (at pH 7.0). Furin-treated particles were later incubated with 6 U of recombinant furin for 3 h at 37 °C. Sodium dodecyl sulfate (SDS) loading buffer and DTT were added to all samples and heated at 95°C for 10 minutes. Samples were separated on NuPAGE Bis-Tris gel (Invitrogen) and transferred on polyvinylidene difluoride membranes (GE). SARS-CoV-2 S was detected using a rabbit polyclonal antibody against the S2 domain (Cat: 40590-T62, Sinobiological) and an AlexaFluor 488 goat anti-rabbit antibody. Bands were detected using the ChemiDoc Imaging software (Bio-Rad) and band intensity was calculated using the analysis tools on Biorad Image Lab 6.1 software to determine the uncleaved to cleaved S ratios.
Chemidoc imaging software
Manufactured by Bio-Rad
Sourced in United States
The ChemiDoc Imaging software is a comprehensive platform for capturing and analyzing images of gel-based and membrane-based assays. It provides a user-friendly interface for acquiring, processing, and quantifying images of various biological samples, including DNA, RNA, and protein.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by GE Healthcare (3 mentions)
Optima max e, by Beckman Coulter (3 mentions)
Dec rvkr cmk, by Bio-Techne (3 mentions)
Image lab 6, by Bio-Rad (3 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
E cadherin, by Proteintech (1 mentions)
Vimentin, by Proteintech (1 mentions)
β catenin, by Proteintech (1 mentions)
C myc, by Proteintech (1 mentions)
β actin, by Proteintech (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Proteome profiler human xl cytokine array, by R&D Systems (1 mentions)
9 protocols using chemidoc imaging software
1
SARS-CoV-2 Spike Protein Cleavage Assay
2
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples of HepG2 and SMMC-7721 cells were homogenized in radioimmunoprecipitation assay (RIPA) buffer containing phenylmethylsulfonyl fluoride (PMSF) for total protein extraction according to the manufacturer’s instructions. Equal amounts of proteins (30 μg per lane) were separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After transferring to Polyvinylidene Fluoride (PVDF) membranes, the membranes were blocked using 5% nonfat milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 2 h and then incubated overnight at 4°C with primary antibodies against PKM2, ODC1, c-myc, E-cadherin, vimentin, β-catenin, and β-actin (Proteintech, Wuhan, China) and p-AKT (Ser473) and p-GSK-3β (Ser9) (CST, USA). The membranes were washed with TBST and incubated with horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibody. Protein bands were visualized using Bio-Rad Chemidoc imaging software (California, USA).
3
Quantification of SARS-CoV-2 Spike Protein Cleavage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 3-mL volume of pseudoparticles was pelleted using a TLA-55 rotor with an Optima-MAX-E ultracentrifuge (Beckman Coulter) for 2 h at 42,000 rpm at 4°C. Untreated particles were resuspended in 30 μL DPBS buffer. Pseudoparticles were generated as described in the pseudoparticle generation section, with the furin inhibitor dec-RVKR-CMK (cat.: 35-011, Tocris) being added during transfection to select wells. For the + furin treated MLVpps, particles were resuspended in 30 μL of furin buffer consistent in 20 mM HEPES, 0.2 mM CaCl2, and 0.2 mM β-mercaptoethanol (at pH 7.0). Furin-treated particles were later incubated with 6 U of recombinant furin for 3 h at 37°C. Sodium dodecyl sulfate (SDS) loading buffer and DTT were added to all samples and heated at 95°C for 10 min. Samples were separated on NuPAGE Bis-Tris gel (Invitrogen) and transferred on polyvinylidene difluoride membranes (GE). SARS-CoV-2 S was detected using a rabbit polyclonal antibody against the S2 domain (cat.: 40590-T62, Sinobiological). Bands were detected using the ChemiDoc Imaging software (Bio-Rad), and band intensity was calculated using the analysis tools on Bio-Rad Image Lab 6.1 software to determine the uncleaved to cleaved S ratios.
4
Profiling 105 Human Secreted Cytokines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of 105 human secreted cytokines in each of the engineered BHPrS1 cells were evaluated using an HXL human cytokine antibody array kit (Cat# ARY022B, R&D Systems, Minneapolis, MN, USA). Human XL Cytokine Arrays were incubated overnight at 4 °C with 500 µL conditioned media, and the procedure was performed according to the manufacturer’s instructions. Following incubation with a detection antibody cocktail, antibody conjugation, and recommended washes, the immunoblots on the membrane were developed with the Chemiluminescent Substrate Reagent Kit (Bio-Rad, Hercules, CA, USA). Signals on each array were detected using ChemiDoc Imaging software (Bio-Rad, Hercules, CA, USA) and signal intensity was quantified using the Fiji plugin in ImageJ software (Version 1.53o) [38 (link)]. The mean signal intensities were subtracted from the median background intensities for background correction. Up (≥1.5-fold) or downregulation (≤0.5-fold) in cytokine secretion were considered significant (p < 0.01) in proteins showing a signal density value >200 pixels.
5
Subcellular Localization of NF-κB in THP-1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
THP-1 cells were treated with BBR overnight followed by LPS treatment for 1 h. After the treatments, cells were lysed in a nuclear extraction (NE-PER) buffer in order to obtain cytosol and nuclear fractions as per manufacturer’s protocol. The total protein in both fractions was measured by Bradford’s method. Cytosolic and nuclear protein fractions (50 μg/lane) were loaded and subjected to SDS-PAGE and then wet transferred onto nitrocellulose membrane (NCM). The membrane was blocked with 15% milk powder in TBS at room temperature for 1 h and then probed with primary anti.rabbit NFp65 antibody (1:1000) overnight at 4 ℃. After incubation, NCM was washed with TBST for 1 h and incubated with anti-rabbit HRP secondary antibody (1:3000). Antibodies of GAPDH (1:1000) or histone (1:1000) were used as internal references for the equal loading of cytosol or nuclear extracts, respectively. Finally, an enhanced ECL reagent was used for blot visualization in a Bio-Rad chemidoc imaging system (USA) using chemidoc imaging software (Bio-Rad, Hercules, CA, USA)
6
Profiling Cytokine Secretion in Plasma
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteome Profiler Human XL Cytokine Array (Cat# ARY022B, R&D Systems, Minneapolis, MN, USA) was used to analyze the expression of 105 human secretory cytokines in patient plasma samples. Nitrocellulose membranes pre-coated with capture antibodies were incubated overnight at 2−8 oC with 200 µl of plasma sample diluted in a buffered protein base according to the manufacturer’s protocol. Subsequently, membranes were incubated with a biotinylated detection antibody cocktail and antibody conjugation with streptavidin-HRP was followed by the recommended washes. The membranes were developed using Chemi Reagent Mix and signal detection was accomplished with ChemiDoc Imaging software (Bio-Rad, Hercules, CA, USA). Signal intensity (densitometry) was quantified using Quick Spots/ HLImage + + software (Ideal Eyes Systems, UT, USA).
7
Analyzing NF-κB and STAT1 Signaling in HaCaT Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HaCaT cells (1 × 106 cells/well) were treated with TNF-α/IFN-γ or with medium alone in the presence or absence of NS or FS for 30 min for the detection of IκB-α, phospho-NF-κB p65, and STAT1. The cells were washed twice with PBS and lysed in a lysis buffer (150 mM NaCl, 50 mM Tris-HCl, 1% Trizol, 0.1% SDS, 0.25% sodium deoxycholate, and 2-mercaptoethanol) containing protease and phosphatase inhibitors. Equal amounts of protein extracts (40 µg) were subjected to 10% SDS-PAGE, and the resultant gel was transferred to a PVDF membrane. The membrane was incubated with TBS-T buffer (pH 7.5, 150 mM NaCl, 50 mM Tris-HCl, and 0.1% Tween 20) containing 5% skim milk for 1 h, followed by incubation at 4 °C overnight with the primary antibodies. Subsequently, the membrane was incubated with the secondary antibodies for 1 h at room temperature. The membrane was washed three times with TBS-T, and color was detected by the PowerOpti-ECL western blotting detection reagent (Bionete, Hwaseong, Korea). The images were captured using the ChemiDoc imaging software (Bio-Rad, Hercules, CA, USA).
8
Quantification of SARS-CoV-2 Spike Protein Cleavage
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 3-mL volume of pseudoparticles was pelleted using a TLA-55 rotor with an Optima-MAX-E ultracentrifuge (Beckman Coulter) for 2 h at 42,000 rpm at 4°C. Untreated particles were resuspended in 30 μL DPBS buffer. Pseudoparticles were generated as described in the pseudoparticle generation section, with the furin inhibitor dec-RVKR-CMK (cat.: 35-011, Tocris) being added during transfection to select wells. For the + furin treated MLVpps, particles were resuspended in 30 μL of furin buffer consistent in 20 mM HEPES, 0.2 mM CaCl2, and 0.2 mM β-mercaptoethanol (at pH 7.0). Furin-treated particles were later incubated with 6 U of recombinant furin for 3 h at 37°C. Sodium dodecyl sulfate (SDS) loading buffer and DTT were added to all samples and heated at 95°C for 10 min. Samples were separated on NuPAGE Bis-Tris gel (Invitrogen) and transferred on polyvinylidene difluoride membranes (GE). SARS-CoV-2 S was detected using a rabbit polyclonal antibody against the S2 domain (cat.: 40590-T62, Sinobiological). Bands were detected using the ChemiDoc Imaging software (Bio-Rad), and band intensity was calculated using the analysis tools on Bio-Rad Image Lab 6.1 software to determine the uncleaved to cleaved S ratios.
9
Phosphokinase Profiling of EFNB Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human phosphokinase antibody array (ARY003C, R&D Systems, Minneapolis, MN, USA) was used to detect the expression of 43 kinase phosphorylation sites on proteins isolated from BHPrS1EV/BHPrS1EFNB1/BHPrS1EFNB2/BHPrS1EFNB3 cells. A total of 400 µg of cell lysate per sample was incubated with antibody array membranes in a multiwell dish overnight and analyzed following the manufacturer’s protocol. Signals were detected using ChemiDoc Imaging software (Bio-Rad, Hercules, CA, USA) and densitometric analysis was carried out using the Fiji plugin in ImageJ software [38 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!