To express proteins in the cell-free system, all inserts were transferred by Gateway LR recombination reaction (Invitrogen) into Gateway compatible modified pTnT vectors (Promega) containing an N-terminal HA or FLAG tag as specified in each case. pDONR207-PIF7 was recombined with the pTnT-HA vector to produce PIF7 fused to HA. The construct containing GI in the pTnT-FLAG vector has already been described (19 (link)). For the in vitro pull-down assays, proteins were co-expressed using TnT® SP6 High-Yield Wheat Germ Protein Expression System (Promega) as per manufacturer’s instructions. Five percent of the reactions (2.5 µl) were used to verify expression of the proteins (input) and the remaining extract was immunoprecipitated as earlier described (47 (link)) using a modified IP buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 1x protease inhibitor cocktail (Roche), 1x Phosphatase Inhibitors I&II (Sigma) and 25 μM MG-132) and the anti-HA 3F10 antibody (Roche).
Phosphatase inhibitors 1 2
Manufactured by Merck Group
Phosphatase Inhibitors I&II are laboratory reagents used to inhibit the activity of phosphatases, enzymes that remove phosphate groups from proteins and other molecules. These inhibitors are commonly used in biochemical research to maintain the phosphorylation state of cellular proteins, which is crucial for various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (2 mentions)
Gateway lr recombination reaction, by Thermo Fisher Scientific (1 mentions)
Anti ha antibody 3f10, by Roche (1 mentions)
Gateway compatible modified ptnt vectors, by Promega (1 mentions)
Tnt sp6 high yield wheat germ protein expression system, by Promega (1 mentions)
Horseradish peroxidase linked secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Nupage, by Thermo Fisher Scientific (1 mentions)
Invitrolon polyvinylidene difluoride membranes, by Thermo Fisher Scientific (1 mentions)
Ecl reagent, by Bio-Rad (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
3 protocols using phosphatase inhibitors 1 2
1
In Vitro Protein Interaction Assay
2
Immunoblotting Protocol for Protein Analysis
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For immunoblotting, cells were lysed in a solution containing 50 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, complete protease inhibitor (Roche) and phosphatase inhibitors I & II (Sigma) and analysed by SDS-PAGE using 4 to 12% Bis-Tris gels (NuPAGE; Invitrogen), followed by electrotransfer onto Invitrolon polyvinylidene difluoride membranes (Invitrogen). Membranes were blocked with 5% (wt/vol) non-fat dry milk and 0.1% (vol/vol) Tween-20 in Tris-buffered saline for 1 hour at room temperature, before being probed with the primary antibody by overnight incubation at 4 °C, followed by incubation for 1 hour at room temperature with a horseradish peroxidase-linked secondary antibody (Santa-Cruz) and detection using ECL reagents (Bio-Rad, Hercules, USA), following the manufacturer’s protocol. Immunoblots were quantified by scanning of films with a calibration strip and analysis by densitometry using Image J (US National Institutes of Health; http://rsb.info.nih.gov/ij ).
3
Transient and Arabidopsis Protein Analysis
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To determine protein levels in transient experiments in N. benthamiana, samples were homogenized with 3 volumes of 2x SDS-PAGE loading buffer and boiled at 95 ˚C for 5 min. Samples were then clarified by centrifugation at room temperature and analyzed by Western blot. For normalization, GFP-HA was used as internal loading control.
For the analysis of PIF7-ECFP-HA protein levels in Arabidopsis seedlings, samples were homogenized with 100 µl of extraction buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 10% glycerol, 2 mM PMSF, 1x protease inhibitor cocktail (Roche), 1x Phosphatase Inhibitors I&II (Sigma) and 50 μM MG-132) and clarified twice by centrifugation at 4 ˚C. Total protein concentration was quantified by DC Protein Assay (Bio-Rad) and 40 ug of each sample was subsequently analyzed by Western blot. ACTIN levels in the samples were used for normalization.
For the analysis of PIF7-ECFP-HA protein levels in Arabidopsis seedlings, samples were homogenized with 100 µl of extraction buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl, 5 mM MgCl2, 0.1% NP-40, 10% glycerol, 2 mM PMSF, 1x protease inhibitor cocktail (Roche), 1x Phosphatase Inhibitors I&II (Sigma) and 50 μM MG-132) and clarified twice by centrifugation at 4 ˚C. Total protein concentration was quantified by DC Protein Assay (Bio-Rad) and 40 ug of each sample was subsequently analyzed by Western blot. ACTIN levels in the samples were used for normalization.
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