Cell extracts were prepared using 2x sample buffer [120 mM Tris-HCl (pH 6.8), 60 mM SDS, 100 mM DTT, 1.75% glycerol, 0.1% bromophenol blue] supplemented with 5 mM MgCl2 and 5 U/ml benzonase. Samples were denatured by heating to 95°C for 5 min. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane that was blocked by incubation in 5% skim milk-containing PBS-0.05% Tween 20, pH 7.4 (PBST) for 1 h at room temperature (RT). The membrane was incubated with a primary antibody in 1% skim milk-containing PBST for either 1 h at RT or overnight at 4°C and subsequently incubated with a secondary antibody conjugated with horseradish peroxidase (HRP) for 1 h at RT. Bound secondary antibodies were detected using the Western Lightning Plus-ECL reagent (PerkinElmer) and signals were visualized by using the Intas ChemoCam Imager 3.2 (Intas).
Chemocam imager 3
Manufactured by Intas
Sourced in Germany, United States
The ChemoCam Imager 3.2 is a compact and versatile laboratory instrument designed for chemical analysis. It utilizes advanced imaging and spectroscopy techniques to provide detailed information about the composition and properties of various samples. The core function of the ChemoCam Imager 3.2 is to capture high-resolution images and analyze the chemical signatures of the samples under investigation.
Automatically generated - may contain errors
Lab products found in correlation
Western lightning plus ecl reagent, by PerkinElmer (6 mentions)
Tween 20, by Carl Roth (3 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Hrp linked goat anti rabbit igg, by Cell Signaling Technology (2 mentions)
Bovine serum albumin (bsa), by AppliChem (1 mentions)
Benzonase, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Tween 20, by Cell Signaling Technology (1 mentions)
Halt phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Fastprep 24 5g instrument, by MP Biomedicals (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
Ecl prime western blotting system, by GE Healthcare (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Goat anti mouse igg, by Merck Group (1 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
Goat anti rabbit igg, by Merck Group (1 mentions)
Dna loading buffer, by Thermo Fisher Scientific (1 mentions)
17 protocols using chemocam imager 3
1
Western Blotting Procedure for Protein Analysis
2
Western Blot Analysis of Viral Proteins
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Samples were denatured in 2x protein sample buffer (200 mM Tris [pH 8.8], 5 mM EDTA, 0.1% Bromophenolblue, 10% sucrose, 3% SDS, 1 mM DTT) and incubated for 5 min at 95°C. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto polyvinylidene difluorid membranes by using a MINI-SDS-PAGE wet-blotting apparatus (Bio-Rad, Munich, Germany). Membranes were blocked with 5% non-fat dry milk in PBS/0.5% Tween-20 (PBST) and incubated with primary antibodies (capsid 1:50; GAPDH 1:1,000; HA 1:1,000; E 1:1,000; prM 1:500; NS1 1:500) by over-night incubation at 4°C or for 1 h at room temperature. After 3 washes with PBST, membranes were incubated with secondary horse radish peroxidase-conjugated antibodies, developed with the Western Lightning Plus-ECL reagent (Perkin Elmer; Waltham, MA) and bands were imaged using an Intas ChemoCam Imager 3.2 (Intas, Göttingen).
3
Western Blot Analysis of HCV Proteins
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Samples were mixed with 5X Laemmli buffer and incubated for 5 min at 95°C. Proteins were separated by electrophoresis into 8% (for NS5A, E2, β-actin and ADRP) or 15% (for core protein) polyacrylamide gels and transferred to a PVDF membrane. Membranes were blocked with PBS containing 0.5% Tween-20 (PBST) supplemented with 3% bovine serum albumin (BSA; Applichem). Primary antibodies [NS5A (1:10,000), Core (C7/50; 1:1,000), ADRP (1:1,000), β-actin (1:20,000) and E2 (3/11; 1:500)] were diluted in PBST containing 1% BSA and membranes were incubated with the antibodies for 1 h at RT or overnight at 4°C. Membranes were washed 3 times with PBST and incubated for 1 h at RT with the corresponding horseradish peroxidase-conjugated secondary antibody. After 3 washes with PBST, bound secondary antibody was detected by using the Western lightning plus-ECL reagent according to the instructions of the manufacturer (PerkinElmer, Waltham, MA). Signals were detected by using the Intas ChemoCam Imager 3.2 (Intas, Göttingen, Germany) and quantified with the LabImage 1D Intas software package.
4
Immunoprecipitation and Western Blotting
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For immunoprecipitation, cells were processed as described for proteome analysis, but captured protein complexes were eluted by using 2× sample buffer (100 mM Tris-HCl [pH 6.8], 4% SDS, 12% β-mercaptoethanol, 20% glycerol, 0.001% bromophenol blue). To prepare lysates for immunoblotting, cells were incubated in 2× lysis buffer (200 mM Tris [pH 8.8], 5 mM EDTA, 0.1% bromophenol blue, 10% sucrose, 3% SDS, 2% β-mercaptoethanol) and incubated for 5 min at 95 °C. Proteins were separated by SDS-polyacrylamide gel electrophoresis and electro-transferred onto PVDF membranes. After blocking of the membranes with 5% nonfat milk, they were incubated overnight at 4 °C with primary antibodies. After washing with 0.5% Tween 20 in PBS, membranes were incubated with secondary horseradish peroxidase-conjugated antibodies for 1 h at room temperature. Membranes were developed by using Western Lightning Plus-ECL reagent (PerkinElmer), and signals were detected by Intas ChemoCam Imager 3.2 (Intas).
5
Western Blot Protein Detection Protocol
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Cell lysates were mixed with 2x sample buffer (120 mM Tris-HCl [pH 6.8], 60 mM SDS, 100 mM DTT, 1.75% glycerol, 0.1% bromophenol blue) and incubated for 15 min at 95°C. Proteins were separated by electrophoresis into SDS - 10% polyacrylamide gels and transferred to a PVDF membrane. Membranes were blocked with PBST supplemented with 5% skim milk. Primary antibodies (see Key Resources Table for detail) were diluted in PBST containing 0.5% skim milk and membranes were incubated with the antibodies overnight at 4°C. After several washings, membranes were incubated for 1 h at room temperature with the corresponding secondary antibody. Signals were detected using the Western lightning plus-ECL reagent (PerkinElmer) and an Intas ChemoCam Imager 3.2 (Intas).
6
Western Blot Analysis of Viral Proteins
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Transfected cells in 6-well plates were washed with PBS and lysed with 100 μl of sample buffer (120 mM Tris-HCl [pH 6,8], 60 mM SDS, 100 mM DTT, 1.75% glycerol, 0.1% bromophenol blue) supplied with 1 μl of benzonase (Milipore: 70746-3) to digest contaminating nucleic acids. Samples were denatured by incubating at 95°C for 3 min. After SDS-PAGE, proteins were blotted onto PVDF (polyvinylidenfluorid) membranes that were blocked with 5% milk in PBS-T (PBS containing 1% Tween) for 1h at RT. Membranes were incubated with primary antibodies, diluted in 3% BSA in PBS, for 1h and washed three times for 10 min each with PBS-T. Horse radish peroxidase (HRP)-conjugated secondary antibodies were diluted in 5% milk in PBS-T and membranes were incubated for 1h at RT. After washing three times with PBS-T for 10 min each, membranes were developed using the Western Lightning Plus-ECL reagent (Perkin Elmer: NEL105001EA). Signals were visualized using a ChemoCam Imager 3.2 (Intas Science Imaging Instruments GmbH, Göttingen, Germany). Signals were quantified using the ImageJ (FiJi) software package (Schindelin et al., 2012 (link)). Values obtained for DENV or ZIKV NS1, NS4B or NS5 were normalized to the one obtained for NS3 of the respective virus. Same amount of lysate (based on equal cell numbers) was loaded onto each lane of the gel.
7
Western Blot Analysis of Pneumolysin
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Concentrated supernatants obtained for the hemolysis assay were separated electrophoretically using a 5% stacking and a 10% running sodium dodecyl sulfate-polyacrylamide gel and were transferred to a polyvinylidene fluoride membrane (Merck). The membranes were blocked for 1 h at room temperature (RT) with 5% skimmed milk powder in Tris-buffered saline (TBS) with 1% Tween®20 (Carl Roth). Afterward, they were incubated with a polyclonal rabbit antiserum raised against Ply (Davids Biotechnologie GmbH, Germany; dilution 1:500 in 1% skimmed milk powder in TBS with 1% Tween®20) [38 (link)] overnight at 4°C, followed by incubation with HRP-linked goat anti-rabbit IgG (Cell Signaling; dilution 1:5,000 in 1% skimmed milk powder in TBS with 1% Tween®20) for 1 h at RT. The membranes were developed with SuperSignal™ West Pico Chemiluminescent Substrate (Thermo Fisher Scientific), and the chemiluminescent signal was detected with ChemoCam Imager 3.2 (Intas, Germany).
8
Western Blotting Protein Analysis
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For protein analysis by Western blotting, whole tissue lysates were prepared in cell extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with protease inhibitor cocktail P8340, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride and Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) using an MP Biomedicals™ FastPrep-24™ 5G Instrument (St. Ana, CA, USA). Crude lysates were cleared by centrifugation and protein amounts were quantified by MicroBC Assay Protein Quantitation Kit (Interchim, Montluçon, France). Equal protein amounts (15 μg) were separated on 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels which were then blotted onto polyvinylidene fluoride membranes. The latter were blocked in 5% (w/v) skimmed milk for 1 h at room temperature, washed and incubated overnight with the respective primary antibodies at 4 °C. After washing, the blots were incubated for 1 h with secondary antibodies against rabbit IgG and washed. All washing steps were performed in tris-buffered saline (TBS)/0.05% (v/v) Tween® 20 (3 × 5 min). Blots were developed using SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) and a ChemoCam Imager 3.2 (Intas, Göttingen, Germany). Densitometry was performed using LabImage 1D version 4.1 (Kapelan Bio-Imaging, Leipzig, Germany).
9
Immunoblotting Analysis of Interferon Regulatory Factors
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Cells were directly lysed in Laemmli sample buffer (0.1 M TRIS [pH 6.8], 30% glycerol, 3% SDS, 7.5% b-mercaptoethanol, 0.06% bromophenol blue) and boiled 5 min at 95 °C for denaturation. Proteins were separated on 10% SDS-PAGEs, transferred onto PVDF membranes (1620177, Bio-Rad) and stained for IRF1, IRF2, IRF3, IRF5, IRF7, IRF9, RIG-I, RELA, and Calnexin (see Table 2 ). Secondary HRP conjugated goat anti-mouse (1:10,000 dilution) or anti-rabbit (1:20,000 dilution) antibodies (Sigma Aldrich) were used to detect the primary antibodies. Clarity Western ECL Blotting Substrates (170-5061, Bio-Rad) or ECL Prime Western Blotting System (RPN2236, GE Healthcare Life sciences) were used to incubate the membranes and chemo luminescence was detected by a high-sensitivity CCD camera (ChemoCam Imager 3.2, INTAS, Germany). Images were taken without binning at 16 bit/pixel. LabImage 1D software (INTAS/KAPELAN, Germany) was used for the quantification of luminescent signals of immunoblots. Signals of the target proteins were normalized to the respective loading controls.
10
Western Blot Analysis of p53 and p21
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Whole-cell lysates were prepared, separated in 11% SDS-PAGE gels, and blotted on Immobilon-PVDF membrane (Merck Millipore, Billerica, MA) as previously described [11] (link). Membranes were incubated with antibodies against p53 (DO-7, BD Biosciences, Franklin Lakes, NJ; 1:1500), p21 (CP36/CP74, Merck Millipore, Billerica, MA; 1:1000), or GAPDH (14C10, Cell Signaling Technology, Danvers, MA; 1:3000). Secondary HRP-conjugated antibodies (Goat-anti mouse IgG and Goat-anti rabbit IgG, Merck Millipore, Billerica, MA; each 1:5000) and ECL chemiluminescent substrate were used for visualization at a ChemoCam Imager 3.2 (INTAS, Göttingen, Germany). Band densities were quantified using QuantityOne 4.6.7 (Bio-Rad, Hercules, CA). For control, always two values were used to reduce the overall scatter of the data. Densities of p53 and p21 bands were normalized to the expression of the housekeeping protein GAPDH and then corrected for the intensity of the total blot.
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