Protein was extracted from yeast cultures and analyzed by western blotting as previously described (43 (link)). Rabbit polyclonal anti-LexA (Invitrogen), rabbit polyclonal anti-Gal4AD (Sigma) and mouse monoclonal anti-Pgk1 (Abcam) were used as primary antibodies with Horseradish peroxidase (HRP)-conjugated goat anti-rabbit (Novex) and anti-mouse IgG (Sigma) as secondary antibodies. Signals were detected by chemiluminescence using a Clarity Western ECL Substrate Kit (BioRad) and images captured using a VersaDoc 4000MP imaging system and Quantity One software (BioRad) or by autoradiography. Images were processed using Adobe Photoshop CS6 and figures prepared using Adobe Illustrator CS6.
Versadoc 4000mp imaging system
Manufactured by Bio-Rad
Sourced in United States
The VersaDoc 4000MP imaging system is a multipurpose imaging system designed for a variety of applications in life science research. It captures high-quality images of samples using different detection methods, including chemiluminescence, fluorescence, and visible light.
Automatically generated - may contain errors
Lab products found in correlation
Supersignal west pico, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Cytiva (4 mentions)
Protease inhibitor cocktail, by Merck Group (4 mentions)
Micromass q tof ultima, by Thermo Fisher Scientific (3 mentions)
Q exactive hybrid quadrupole orbitrap, by Thermo Fisher Scientific (3 mentions)
Cary 300 bio, by Agilent Technologies (3 mentions)
Trans blot turbo transfer system, by Bio-Rad (3 mentions)
Quantity one software, by Bio-Rad (2 mentions)
Clarity western ecl substrate kit, by Bio-Rad (2 mentions)
Chemidoc mp imaging system, by Bio-Rad (2 mentions)
Anti β actin, by Abcam (2 mentions)
Polyacrylamide ready gel, by Bio-Rad (2 mentions)
Donkey anti goat igg hrp, by Thermo Fisher Scientific (2 mentions)
Tris hcl denaturing polyacrylamide ready gel, by Bio-Rad (2 mentions)
Goat anti rabbit igg hrp, by Thermo Fisher Scientific (2 mentions)
Goat anti β actin, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal anti pgk1, by Abcam (1 mentions)
18 protocols using versadoc 4000mp imaging system
1
Western Blot Analysis of Yeast Proteins
2
Yeast Protein Extraction and Western Blot Analysis
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Protein was extracted from yeast cultures using alkali lysis (12 (link)) and analyzed by western blotting as previously described (15 (link)). Antibodies used were rabbit polyclonal anti-Rep1 and anti-Rep2 (15 (link)), rabbit polyclonal anti-LexA (Pierce), mouse monoclonal anti-PGK (Molecular Probes), horseradish peroxidase-conjugated goat anti-rabbit (KPL) and Dylight 549 or 649-conjugated goat anti-mouse IgG (Rockland). Signals were detected by chemiluminescence using a Clarity Western ECL Substrate kit (BioRad) or by fluorescence and imaged using a VersaDoc 4000 MP imaging system with Quantity One software (BioRad) or by autoradiography.
3
Quantifying Plant Fluorescence Imaging
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Fluorescence was monitored in whole plants using a VersaDoc 4000 MP Imaging System (Bio-Rad, Hercules, CA, USA) set with the following parameters: Light mode: LED epi, color: Blue, filter name: 530 BP, gain setting: 1×, and exposure time: 3–120 s.
For microscopy analysis, 2 leaf disks were mounted in water between a slide and cover glass with the upper epidermis forward with a 10× objective. Fluorescence microscopy analysis was performed using a BX53 microscope (Olympus, Shinjuku, Tokyo, Japan) with a GFP-specific filter. Laser-scanning confocal microscopy was performed at the Laboratory of Electron and Confocal Microscopy (Faculty of Biology, Adam Mickiewicz University, Poznań, Poland).
Additionally, fluorescence was measured in a crude extract prepared from plants verified for fluorescence by a DTX 880 Multimode Detector (Beckman Coulter, Brea, CA, USA). For this experiment, 3 disks (5 mm in diameter) from each leaf were sampled and homogenized in 100 µL of sterile water, followed by centrifugation to remove the plant debris. The resulting supernatant was taken for analyses. Fluorescence was measured in a black 96-well plate with a clear bottom using 485/535 nm (excitation/emission) filters.
For microscopy analysis, 2 leaf disks were mounted in water between a slide and cover glass with the upper epidermis forward with a 10× objective. Fluorescence microscopy analysis was performed using a BX53 microscope (Olympus, Shinjuku, Tokyo, Japan) with a GFP-specific filter. Laser-scanning confocal microscopy was performed at the Laboratory of Electron and Confocal Microscopy (Faculty of Biology, Adam Mickiewicz University, Poznań, Poland).
Additionally, fluorescence was measured in a crude extract prepared from plants verified for fluorescence by a DTX 880 Multimode Detector (Beckman Coulter, Brea, CA, USA). For this experiment, 3 disks (5 mm in diameter) from each leaf were sampled and homogenized in 100 µL of sterile water, followed by centrifugation to remove the plant debris. The resulting supernatant was taken for analyses. Fluorescence was measured in a black 96-well plate with a clear bottom using 485/535 nm (excitation/emission) filters.
4
Western Blot Analysis of Apoptosis Regulators
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Cells were completely lysed in 200 l of the lysis buffer (Takara, Dalian, China) and then centrifuged at 8000g for 5 min. Proteins were separated by 12% SDS-PAGE, and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked by 5% skimmed milk in TBS for 2 h. After washed three times by TBS containing 0.1% Tween-20 (TBST), the PVDF membranes were incubated with anti-Bax (Cell Signaing Technology, Danvers, MA, USA), anti-Bcl2 (Cell Signaing Technology), anti-SMAD7 (Cell Signaing Technology), anti-β-actin (Cell Signaing Technology) overnight at 4 °C, respectively. After washed with TBST, the PVDF membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology). Lastly, the PVDF membranes were exposed to ECL Western Blotting Substrate (Solarbio, Beijing, China) for 5 min and were quantified using VersaDoc 4000MP imaging system (Bio-Rad).
5
Analytical Characterization of Compounds
6
SDS-PAGE Analysis of SEC Fractions
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An equal volume (40 µL) from each of the collected SEC fractions was boiled in reducing buffer and subjected to electrophoresis on precast Mini-Protean TGX 4–20% gradient gels (Bio-Rad Laboratory). Gels were incubated with PageBlue Protein Staining Solution (Thermo Scientific, Waltham, MA, USA) for 2 h with gentle agitation at RT and then washed three times in dH2O before being detected with a VersaDoc 4000 MP imaging system (Bio-Rad Laboratories, Hercules, CA, USA).
7
Western Blot Analysis of Protein Expression
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Proteins were isolated using standard RIPA buffer protocols. 15 µg of the proteins were separated on precast Mini-Protean 4-15% TGX gels at 250 Volts (Bio-Rad Laboratories, Hercules, CA, USA). Afterwards, the proteins were transferred on Trans-Blot Turbo 0,2 µm PVDF membranes via the Trans-Blot Turbo Transfer System and the preinstalled mixed molecular weight program (Biorad). The membranes were blocked with a 3% non-fatty instant milk PBS/0.05% Tween-20 solution under constant shaking. Antibody detection was performed using antibodies against p53 (BioLegend, San Diego, CA, USA), GAPDH (ProteinTech Europe, Manchester, UK) and CAIX (Novus Biologicals LLC, Littleton, CO, USA). HRP-conjugated secondary antibodies were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Bands were visualized by using the WesternBright Chemiluminescence Substrate Sirius (Biozym Scientific GmbH, Oldendorf, Germany) and the VersaDoc 4000MP Imaging System (Biorad).
8
Extracellular Vesicle Protein Analysis by Western Blot
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Forty microliters of each SEC fraction were loaded and separated on Mini-Protean TGX precast 4–20% gels (Bio-Rad Laboratory), and proteins were blotted onto PVDF membranes using a Trans-Blot Turbo Transfer system (Bio-Rad Laboratory). Membranes were blocked with 5% non-fat dry milk in TBS containing 0.01% Tween-20 (TBST). Membranes were incubated with the following primary antibodies: CD81 (1:500 dilution; clone H-121, sc-9158, Santa Cruz Biotechnology, Santa Cruz, CA), TSG-101 (1:500 dilution; clone 4A10, ab83, Abcam, Cambridge, UK), flotillin-1 (1:1000 dilution; clone H-104, sc-25506, Santa Cruz Biotechnology), and Apo-A (1:1000 dilution; clone FL-267, sc-30089, Santa Cruz Biotechnology), diluted in TBST overnight at 4 °C. Membranes were washed three times before being incubated with the following secondary antibodies diluted in TBST; donkey anti-rabbit IgG HRP-linked F(ab′)2 fragment (1:10,000 dilution; NA9340V), and sheep anti-mouse IgG HRP-linked F(ab′)2 fragment (1:10,000 dilutions; NA9310V) (both from GE Healthcare, Buckinghamshire, UK). Blots were visualised with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Scientific) and a VersaDoc 4000 MP imaging system (Bio-Rad Laboratory) with Quantity One software.
9
Characterization of Organic Compounds
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All reagents were purchased from Aldrich or Fisher Scientific and were of the highest purity commercially available. UV spectra were obtained with a Varian Cary 300 Bio UV-visible spectrophotometer. HPLC was performed on a Dionex Ultimate 3000 HPLC system equipped with a diode array detector using Macherey-Nagel C18 reverse-phase column. NMR spectra were acquired on a Bruker AVANCE III 400 MHz high-field NMR spectrometer and the data were processed using Topspin software. HRMS spectra were acquired with either a Waters Micromass Q-tof Ultima or a Thermo Scientific Q-Exactive hybrid Quadrupole Orbitrap. Fluorescence scanning was performed on a Biorad Versa Doc 4000 MP Imaging System or a Biorad ChemiDoc MP imaging system.
10
Antibody Characterization for PI3K Subunits
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