Two days after agroinfiltration, tobacco leaf tissue was frozen in liquid nitrogen and ground in 2× Laemmli buffer with 0.2 M dithiothreitol. The protein samples were boiled for 5 min, centrifuged to pellet leaf debris, then separated by sodium dodecylsulphate–polyacrylamide gel electrophoresis and transferred by electroblotting to a nitrocellulose membrane. The membrane was blocked in 5% skim milk then probed with anti-GFP (7.1 and 13.1; Roche, Penzberg, Germany), followed by sheep anti-mouse IgG antibody conjugated to horseradish peroxidase. Labelling was detected using the Amersham ECL Western Blotting Detection Reagent and the ImageQuant LAS 4000 CCD camera system (GE Healthcare Life Sciences, Little Chalfont, UK).
Imagequant las 4000 ccd camera system
Manufactured by GE Healthcare
Sourced in United Kingdom
The ImageQuant LAS 4000 is a CCD camera system designed for quantitative imaging of gel-based and membrane-based assays. It captures high-resolution images and provides accurate and reproducible quantification of various biomolecules, including proteins, nucleic acids, and small molecules.
Automatically generated - may contain errors
Lab products found in correlation
Crescendo chemiluminescent substrate, by Merck Group (3 mentions)
1.5mm cassettes, by Thermo Fisher Scientific (2 mentions)
Xcell surelock mini cell system, by Thermo Fisher Scientific (2 mentions)
Hybond c nitrocellulose membrane, by GE Healthcare (2 mentions)
Anti gfp 7 1 and 13, by Roche (1 mentions)
Ecl western blotting detection reagent, by GE Healthcare (1 mentions)
Nupage gel, by Thermo Fisher Scientific (1 mentions)
Ap conjugate substrate kit, by Bio-Rad (1 mentions)
Enhanced chemiluminescence substrate, by Merck Group (1 mentions)
Gfp trap beads, by Proteintech (1 mentions)
6 protocols using imagequant las 4000 ccd camera system
1
Western Blot Analysis of GFP in Tobacco Leaves
2
Western Blot Analysis of Cellular Proteins
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Whole cell lysates were subjected to electrophoresis in 4–12% NuPAGE gel (ThermoFisher Scientific). Western blots were performed with primary antibodies listed below; ACTB (Sigma-Aldrich, France, #A1978), NQO1 (Abcam, Cambridge, UK, #ab34173), GCLC (Abcam, #ab41463). Anti-Mouse IgG (whole molecule)-Alkaline Phosphatase (Simga-Aldrich, #A3562) was used as secondary antibody for ACTB. Anti-Rabbit IgG, HRP conjugated (Merck, #NA934VS) was used as a secondary antibody for NQO1 and GCLC. EC Prime Western Blotting System (Merck, Darmstadt, Germany) and AP Conjugate Substrate Kit (BioRad, Calfornia, US) were used for detection. Chemiluminescence images were recorded with an ImageQuant LAS 4000 CCD camera system (GE Healthcare).
3
Aptamer-Based Protein Detection in SDS-PAGE
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Proteins (20 µg) were fractionated in 12.5% SDS-PAGE gels (120 V, 45 min, RT) and electro-transferred for 1 h onto a preactivated polyvinylidene difluoride membrane at 4 °C and 400 mA. The membrane was washed (2×, 5 min) with phosphate-buffered saline, pH 7.4 (PBS), and blocked under orbital stirring (50 rpm) at RT for 1 h with 5% (w/v) skim milk powder in PBS containing 1% Triton X-100, washed again (3×, 5 min), and the aptamers labeled at their 5’-ends with 6FAM (λex/em: 495/520 nm) or with biotin, were added at a final concentration of 0.9 µM and incubated overnight. After washing (3×, PBS), 6FAM fluorescence was measured in an ImageQuant™ LAS 4000 CCD camera system (GE Healthcare) using epi-illumination and a Y515 filter. Biotin-labeled aptamers were revealed after incubation with horseradish peroxidase-conjugated streptavidin (Merck KGaA) and reacted with enhanced chemiluminescence substrate (Merck KGaA) prior to detection in an ImageQuant™ LAS 4000 CCD camera system.
4
SDS-PAGE and Western Blot for Protein Analysis
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SDS - polyacrylamide gel electrophoresis (PAGE) and Western Blotting samples were denatured at 94°C for 5 minutes in 3 x SDS sample buffer (150mM Tris pH 8, 6% SDS, 0.3M DTT, 0.3% Bromophenol Blue, 30% glycerol). Polyacrylamide gels were prepared using 10% running gels and 5% stacking gels in Novex 1.5mm Cassettes and run using the Novex XCell SureLock Mini-Cell system. Gels were transferred onto Hybond-C nitrocellulose membrane (GE Healthcare). Membranes were blocked in 4% milk for 1 h and incubated overnight at 4°C with shaking in the appropriate primary antibody against Nischarin (1:500, BD Biosciences, Cat No: 558262), GFP (1:500, Santacruz, Cat No: sc-8334), V5 (1:2000, Invitrogen, Cat. No: R960-25), GLT (1:500, Alomone). HRP-conjugated secondary antibodies were from Rockland (1:10,000). Bands were visualised using Crescendo Chemiluminescent substrate (Millipore) together with an ImageQuant LAS 4000 CCD camera system (GE Healthcare).
5
Co-immunoprecipitation and Western Blotting
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Co-immunoprecipitation experiments were made in lysis buffer (50 mM Tris, pH 7.5, 0.5% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 μg/ml antipain, pepstatin, and leupeptin) using GFP trap beads (Chromotek) on COS cells lysate. SDS–polyacrylamide gel electrophoresis (PAGE) and Western Blotting were carried out as previously described (Norkett et al., 2016 (link)). HRP-conjugated secondary antibodies were from Rockland (1:10,000). Bands were visualized using Crescendo Chemiluminescent substrate (Millipore) together with an ImageQuant LAS 4000 CCD camera system (GE Healthcare).
6
SDS-PAGE and Western Blotting Protocol
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SDS -polyacrylamide gel electrophoresis (PAGE) and Western Blotting samples were denatured at 94°C for 5 minutes in 3 x SDS sample buffer (150mM Tris pH 8, 6% SDS, 0.3M DTT, 0.3% Bromophenol Blue, 30% glycerol). Polyacrylamide gels were prepared using 10% running gels and 5% stacking gels in Novex 1.5mm Cassettes and run using the Novex XCell SureLock Mini-Cell system. Gels were transferred onto Hybond-C nitrocellulose membrane (GE Healthcare). Membranes were blocked in 4% milk for 1 hour and incubated overnight at 4°C with shaking in the appropriate primary antibody against Nischarin (1:500, BD Biosciences, Cat No: 558262), GFP (1:500, Santacruz, Cat No: sc-8334), V5 (1:2000, Invitrogen, Cat. No: R960-25), GLT (1:500, Alomone). HRP-conjugated secondary antibodies were from Rockland (1:10,000). Bands were visualised using Crescendo Chemiluminescent substrate (Millipore) together with an ImageQuant LAS 4000 CCD camera system (GE Healthcare).
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