Briefly, total cell lysates of control and Sdc-1 siRNA transfected SUM-149 cells were prepared as described before [3 (link)]. Protein concentration was determined using Bradford assay and 50 ug protein per lane were electrophoresed on 10% SDS-PAGE and electrotransferred into nitrocellulose membrane (Millipore, Germany). After blocking with 5% BSA in tris- buffered saline with 0.1% tween (TBST) for 1 hour the membrane was probed with 2 ug/mL primary antibody against DLL4 (Santa Cruz Biotech, CA, USA) overnight at 4 °C. On the next day, the membrane was incubated with 1 ug/mL anti-mouse secondary antibody conjugated with horseradish peroxidase (Santa Cruz Biotech, CA, USA) for 1 hour at room temperature. The immunoreactivity was visualized by Enhanced Chemiluminescence (ECL) reaction and BioSpectrum 815 Imaging System (Analytik Jena, USA). β-actin (Santa Cruz Biotech) was used as a loading control.
Biospectrum 815 imaging system
Manufactured by Analytik Jena
Sourced in United States
The Biospectrum 815 Imaging System is a versatile laboratory instrument designed for high-performance imaging applications. It features a sensitive CCD camera and advanced optics to capture detailed images of a variety of samples, including gels, blots, and cell cultures.
Automatically generated - may contain errors
Lab products found in correlation
Visionworks software, by Analytik Jena (2 mentions)
Amersham ecl western blotting detection reagent, by GE Healthcare (2 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Visionworks acquisition and analysis software, by Analytik Jena (1 mentions)
Ecl prime chemiluminescence system, by GE Healthcare (1 mentions)
Pierce bca protein assay, by Thermo Fisher Scientific (1 mentions)
La taq dna polymerase, by Takara Bio (1 mentions)
Goscript reverse transcription system, by Promega (1 mentions)
Mouse anti nf κb, by Cell Signaling Technology (1 mentions)
Mouse anti stat3, by Cell Signaling Technology (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
6 protocols using biospectrum 815 imaging system
1
Sdc-1 Regulation of DLL4 Expression
2
Western Blot Protein Detection Protocol
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The WB was performed according to standard WB protocol as described previously [34 (link)]. Briefly, the tissue samples were lysed on an ice-cold RIPA buffer supplemented with a protease and phosphatase inhibitors cocktail and briefly sonicated on ice. Protein concentrations were measured using Pierce® BCA protein assay (Thermo Scientific, Rockford, IL). Equal amounts of proteins were loaded onto SDS-PAGE gels and transferred into nitrocellulose membranes. After blocking, the membranes were incubated with primary antibodies overnight at 4 °C followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The immuno-reactive bands were visualized using the ECL prime chemiluminescence system (GE Healthcare Life Sciences, Marlborough, MA) and the images were captured using the BioSpectrum® 815 imaging system (Analytik Jena, Upland, CA). Densitometry analysis was performed using the VisionWorks® acquisition and analysis software (Analytic Jena).
3
Western Blot Analysis of Tumor Cell Lines
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Human and canine TCC cells were plated in 10-cm tissue culture dishes at a density of 2×106 cells/dish in complete media and allowed to attach for 24 hours. The cells were then treated with or without serum, with drugs in a dose-dependent manner alone, or in combinations for 24 hours as mentioned in figure legends. After treatments, the cells were lysed in ice-cold RIPA buffer supplemented with protease and phosphatase inhibitors cocktail and kept at −80°C until Western blot (WB) analyses were performed. Protein concentrations were measured using the BCA protein assay. Equal amounts of proteins were loaded onto sodium dodecyl sulfate polyacrylamide gel electrophoresis gels and transferred to a nitrocellulose membrane. After blocking, the membranes were incubated with primary antibodies overnight at 4°C. Next day, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:3,000 dilution) for 45 minutes at room temperature, and immunoreactive bands were visualized using the chemiluminescence system using enhanced chemiluminescence reagents. The images were captured using the BioSpectrum® 815 Imaging System (UVP, Upland, CA, USA).
4
SARS-CoV-2 Genome Amplification and Sequencing
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A total of nine overlapping sub-genomic fragments that spanned the complete genomic region were amplified using nine pairs of primers (Additional file 2 : Table S2) [29 ] by RT-PCR. First, viral RNA was reverse transcribed using the GoScript Reverse Transcription System (Promega, WI, USA) following the manufacturer’s instructions. The RT reaction was conducted under the following conditions: 25 °C for 5 min, followed by 1 h at 42 °C, then 72 °C for 15 min. Next, the PCR was carried out in a reaction mixture of 50 μL containing 5 μL of 10 × La Taq Buffer (Mg2+ Plus), 8 μL of dNTP mixture (2.5 mM), 2 μM of forward and backward primers, and one unit of high fidelity La Taq DNA polymerase (TaKaRa, Dalian, China). PCR amplification was performed under the following parameters [29 ]: 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 2 min and final extension at 72 °C for 5 min. The PCR results were analyzed by 1% agarose gel electrophoresis and visualized by a biospectrum® 815 imaging system (UVP, USA). Finally, the PCR products were used as templates for bi-directional DNA sequencing by the Sanger dideoxy sequencing method (Invitrogen Ltd., Shanghai).
5
Western Blot Analysis of Immune Signaling
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Cells were treated with 1 μg/mL LPS and 0.6 μg/mL DMGF for 10 h and then lysed in RIPA lysis buffer. The protein concentration of the cell lysate was estimated with the Bradford protein assay using BSA as the standard. Total proteins (50 μg) were separated by SDS-PAGE using a 10% polyacrylamide gel and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk in phosphate-buffered saline with Tween 20 (0.05% v/v Tween-20 in PBS, pH 7.2) for 1 h. The membranes were then incubated with primary antibodies such as anti-mouse NF-κB (1:1000, Cell Signaling), anti-mouse IκB (1:1000, Cell Signaling) and anti-mouse ERα antibodies (1:1000, Bioss) at 4 °C overnight, followed by incubation with a horseradish peroxide-linked secondary antibody (1:10,000). The protein bands were visualized using the Amersham ECL Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK). The intensity of the chemiluminescence signal was quantified using the Biospectrum 815 Imaging System and Vision Works Software (UVP, Upland, CA, USA).
6
Western Blot Analysis of Splenocyte Proteins
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Splenocytes and splenic T cells were respectively treated with CRA for 72 h and 24 h and then lysed in RIPA lysis buffer. The protein concentration of the cell lysate was estimated with the Bradford protein assay using BSA as the standard. Total proteins (50 μg) were separated by SDS-PAGE using a 10% polyacrylamide gel and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk in phosphate buffered saline with Tween 20 (0.05% v/v Tween 20 in PBS, pH 7.2) for 1 h. The membranes were then incubated with primary antibodies such as anti-mouse STAT3 (1:1000, Cell Signaling), pSTAT3 (1:1000, Cell Signaling), anti-mouse Blimp-1 (1:1000, Cell Signaling) and anti-mouse AID (1:1000, Cell Signaling) at 4 °C overnight, followed by incubation with a horseradish peroxide-linked secondary antibody (1:10,000). The protein bands were visualized using the Amersham ECL Western Blotting Detection Reagents (GE Healthcare, Buckinghamshire, UK). The intensity of the chemiluminescence signal was quantified using the Biospectrum 815 Imaging System and Vision Works Software (UVP, Upland, CA).
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