Plant protease inhibitor

Manufactured by Merck Group

Sourced in United States

Plant protease inhibitor is a laboratory product that functions to inhibit the activity of plant-derived proteases. It is commonly used in various research applications involving the study of plant-based biological systems.

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Lab products found in correlation

Nebnext ultra dna library prep kit, by New England Biolabs (2 mentions) Ab9050, by Abcam (1 mentions) Nextseq 500 platform, by New England Biolabs (1 mentions) Nextseq 500, by Illumina (1 mentions) Ab1791, by Abcam (1 mentions) Ab18413, by Abcam (1 mentions) Ab5095, by Abcam (1 mentions) Ab5131, by Abcam (1 mentions) Phosphatase inhibitor, by Merck Group (1 mentions) Anti flag antibody, by Merck Group (1 mentions) Anti ha antibody, by Merck Group (1 mentions) Ab109126, by Abcam (1 mentions) Ecl reagent, by Cytiva (1 mentions) Anti flag agarose, by Merck Group (1 mentions) Anti flag hrp, by Merck Group (1 mentions)

Close spelling variants of this product

Protease inhibitors (2755 citations) Plant protease inhibitor cocktail (53 citations) Protease inhibitor cocktail for plant cell and tissue extracts (9 citations) Protease inhibitor cocktail for plant cell extracts (4 citations) Plant protease inhibitor mixture (3 citations) Protease inhibitor for plant extracts (2 citations) Plant‐specific protease inhibitor cocktail (2 citations) Plant cell protease inhibitor (2 citations) Protease inhibitor cocktail for plant cells (2 citations)

10 protocols using plant protease inhibitor

Chromatin Immunoprecipitation Sequencing Protocol

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Nuclei were isolated from cross-linked samples described as previously ^{47 (link)} and were then resuspended in nuclei lysis buffer (50mM Tris-HCl pH8, 10mM EDTA, 1% SDS, 1mM PMSF, 1% Plant Protease Inhibitors (Sigma)). After fragmentation using a Covaris S200, the chromatin samples were diluted with ChIP dilution buffer (1.1% Triton X-100, 1.2mM EDTA, 16.7mM Tris-HCl pH8.0, 167mM NaCl, 1mM PMSF, 1% Plant Protease Inhibitors (Sigma)). Diluted chromatin samples were subjected to immunoprecipitation with antibodies (anti-FPA; anti-RNA polymerase II CTD repeat YSPTSPS antibody (8WG16), Abacm ab817; and control IgG Abcam ab18413) described as above.
Native histone ChIP was largely performed as described previously ^{48 (link)}, with anti-Histone H3 Abcam ab1791, anti-Histone H3 (tri methyl K36) Abcam ab9050, anti-Histone H3 (tri methyl K4) Millipore 17-614, and anti-Histone H3 (di methyl K4) Millipore 17-677. ChIP libraries were prepared using NEBNext® Ultra™ DNA Library Prep kit (New England Biolabs) and sequenced on the NextSeq 500 platform at Center of Genomics and Bioinformatics, Indiana University.

Yu X., Martin P.G., Zhang Y., Trinidad J.C., Xu F., Huang J., Thum K.E., Li K., Zhao S., Gu Y., Wang X, & Michaels S.D. (2021). The BORDER family of negative transcription elongation factors regulate flowering time in Arabidopsis. Current biology : CB, 31(23), 5377-5384.e5.

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Chromatin Immunoprecipitation and Sequencing

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Nuclei were isolated from cross-linked samples as described previously^{50 (link)} and resuspended in nuclei lysis buffer (50 mM Tris-HCl pH8, 10 mM EDTA, 1% sodium dodecyl sulfate (SDS), 1 mM phenylmethanesulfonylfluoride (PMSF), 1% Plant Protease Inhibitors from Sigma). After fragmentation using a Covaris S200, the chromatin samples were diluted with ChIP dilution buffer (final concentration: 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8.0, 150 mM NaCl, 1 mM PMSF, 0.1% SDS, 1% Plant Protease Inhibitors, Sigma). The diluted chromatin samples were subjected to immunoprecipitation with antibodies (anti-MYC tag, clone 4A6, Millipore 05–724 (30 μg); Anti-RNA polymerase II CTD repeat YSPTSPS antibody [8WG16] Abacm ab817 (20 μg); Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S2) antibody, Abcam ab5095 (30 μg); Anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) antibody Abcam ab5131 (30 μg); and control IgG Abcam ab18413 (20 μg)).
Native histone ChIP was performed as described previously^{51 (link)} using anti-Histone H3 Abcam ab1791 (10 μg).
The ChIP libraries were prepared using the NEBNext® Ultra™ DNA Library Prep Kit (New England Biolabs) and then sequenced on a NextSeq 500 (Illumina) at the Center of Genomics and Bioinformatics, Indiana University. All high-throughput sequencing data and corresponding experimental details are available in GEO SuperSeries GSE112443.

Yu X., Martin P.G, & Michaels S.D. (2019). BORDER proteins protect expression of neighboring genes by promoting 3′ Pol II pausing in plants. Nature Communications, 10, 4359.

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Fractionation of Protein Extracts

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For the separation of soluble and insoluble (with protein aggregates) fractions, native protein extracts were obtained using a buffer containing 100 mM Tris‐HCl pH 7.9, 10 mM MgCl₂, 1% (v/v) glycerol, and 1X plant protease inhibitor (Sigma). After centrifugation for 10 min at 10,000 ×g, the supernatant was collected as the soluble fraction. The pellet was washed with fresh buffer and centrifuged again. The obtained pellet fraction was then resuspended in denaturing TKMES buffer and centrifuged again to collect the supernatant as the insoluble fraction. Protein concentration from each fraction was determined with Pierce Coomassie Plus (Bradford) Protein‐Assay (Thermo Scientific).

Llamas E., Torres‐Montilla S., Lee H.J., Barja M.V., Schlimgen E., Dunken N., Wagle P., Werr W., Zuccaro A., Rodríguez‐Concepción M, & Vilchez D. (2021). The intrinsic chaperone network of Arabidopsis stem cells confers protection against proteotoxic stress. Aging Cell, 20(8), e13446.

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Peptide Extraction from Mung Bean

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Peptides in mung bean seeds and sprouts were extracted as previously described with some modification [23 (link)]. The samples were quickly grounded in liquid nitrogen using a dry ice-cooled mortar and pestle, 5 g of bean powder was then dissolved in extraction buffer containing 1% TFA and 1% plant protease inhibitor (Sigma-Aldrich, MO, USA). Mixed samples were homogenized at 4 °C for 1 h and then sonicated with five short bursts of 6 s followed by intervals of 8 s for cooling on the ice. After that, samples were centrifuged at 10,000g for 20 min at 4 °C and filtered in an Amicon Ultracel 10 kDa Molecular weight (Mw) cut-off centrifuge filter tube (Millipore, MA, USA) by centrifugation (4 °C, 8000g) to remove peptides larger than 10 kDa. The desalting process, mass spectrometry conditions, and the data analysis were the same as the above proteomics analysis, except the peptide separation time was 60 min. Finally, the relative intensity of the peptide was obtained.

Yu W., Zhang G., Wang W., Jiang C, & Cao L. (2020). Identification and comparison of proteomic and peptide profiles of mung bean seeds and sprouts. BMC Chemistry, 14(1), 46.

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CMV Agrotransformation and Protein Extraction

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Throughout this study, we used Q strain of CMV (Q-CMV) [12 (link)] and its sat-RNA (Qsat-RNA) [3 (link)]. Characteristic features of Agrobacterium-based T-DNA constructs of the three genomic RNAs of Q-CMV and Qsat-RNA are as previously described [12 (link), 13 (link)]. Wild-type Nicotiana benthamiana leaves were infiltrated with CMV agrocultures [14 ] [15 (link)]. Either healthy or four days post infiltrated (dpi) N. benthamiana leaves with CMV agrotransformants were used to prepare the total protein extract. Briefly, leaves were ground in liquid nitrogen, and total protein was extracted in 3 volumes of extraction buffer (20 mM Tris-Cl [pH 7.5], 300 mM NaCl, 5 mM MgCl₂, 5 mM DTT, 1% plant protease inhibitor [Sigma, USA]). The liquid extract was centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected for subsequent experiments.

Chaturvedi S, & Rao A.L. (2017). Riboproteomics: A versatile approach for the identification of host protein interaction network in plant pathogenic noncoding RNAs. PLoS ONE, 12(10), e0186703.

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Transient Co-expression and Co-immunoprecipitation of Plant Proteins

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Proteins were transiently co-expressed by Agrobacterium infiltration in leaves of N. benthamiana. C. annuum eEF1A and eEF1Bβ were expressed using HA-tagged pEarlyGate (pEG) 201 vector [34 (link)], and the CMV 2b and PVX TGBp1 proteins were expressed using FLAG-tagged pEG202 vector [34 (link)], respectively. Co-IP assays were performed as described previously [35 (link)]. Briefly, leaves were harvested 2 d after infiltration, and total protein was extracted with an extraction buffer [GTEN: 10% glycerol, 25 mM Tris (pH 7.5), 1 mM EDTA, 150 mM NaCl, 10 mM DTT, 0.1% Triton X-100, 1X plant protease inhibitor (Sigma-Aldrich), 1X phosphatase inhibitor (Sigma), and 2% w/v polyvinypolylpyrrolidone]. Protein extracts were incubated with HA tag antibody-agarose beads for IP from 6 h to overnight. Finally, beads were collected and washed six times with an IP buffer (GTEN containing 0.15% Nonidet P-40 and 1 mM DTT). The bead-bound proteins were eluted with 5 × SDS sample buffer [10% w/v SDS, 10 mM beta-mercaptoethanol, 20% v/v glycerol, 0.2M Tris-HCl (pH 6.8), 0.05% w/v bromophenol blue] and denatured by boiling at 95°C for 5 minutes. The denatured proteins were separated via 12% SDS-PAGE and immunoblotted with anti-HA antibody (Sigma) and anti-FLAG antibody (Sigma).

Hwang J., Lee S., Lee J.H., Kang W.H., Kang J.H., Kang M.Y., Oh C.S, & Kang B.C. (2015). Plant Translation Elongation Factor 1Bβ Facilitates Potato Virus X (PVX) Infection and Interacts with PVX Triple Gene Block Protein 1. PLoS ONE, 10(5), e0128014.

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Arabidopsis Seedling Protein Extraction

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Whole Arabidopsis seedlings were frozen in liquid nitrogen and ground to powder using a mortar and pestle. The frozen powder was collected in lysis buffer (50 mM Tris‐ HCl [pH 7.5], 1 mM dithiothreitol and 10% glycerol supplemented with 1X plant protease inhibitor [Sigma]). After centrifugation 10,000 ×g for 10 min at 4°C, the supernatant was collected and protein concentration was determined. 90 µg of total protein was run on a 3–13% gel in deep blue cathode buffer (50 mM Tricine, 7.5 mM Imidazole and 0.02% Coomassie G250) at 4°C for 3 h at 100 V and then exchange deep blue cathode buffer to slightly blue cathode buffer (50 mM Tricine, 7.5 mM Imidazole, and 0.002% Coomassie G250) and run at 100 V overnight. Proteins were then transferred to a polyvinylidene difluoride membrane at 400 mV for 3 h by semi‐dry blotting. For loading control, the membrane was stained with Ponceau S. Western blot analysis was performed with a monoclonal antibody against CCT1 [1:1,000] (Abcam, ab109126).

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Immunoblot Analysis of HDA6 in Plant Leaf Tissue

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Approximately 300 mg of 14-day-old leaf tissue was ground in liquid nitrogen and suspended in 1 mL of phosphate buffered saline, pH 7.4,1mM PMSF and 1% Plant Protease Inhibitors (Sigma, St. Louis). After 10 min at room temperature, with mixing by inversion, extracts were subjected to centrifugation 2 min at 800 x g. The supernatant was filtered through Miracloth and 60 μg total protein, determined using a Coomassie blue dye binding assay, then subjected to electrophoresis on a 7.5% SDS-PAGE gel and transferred to nitrocellulose. The blot was incubated overnight with anti-HDA6 antisera (raised in rabbits against the C-terminal peptide CDEMDDDNPEPDVNPPSS), followed by anti-Rabbit-HRP for 1 h and film detection using ECL reagent (Amersham).

Blevins T., Pontvianne F., Cocklin R., Podicheti R., Chandrasekhara C., Yerneni S., Braun C., Lee B., Rusch D., Mockaitis K., Tang H, & Pikaard C.S. (2014). A two-step process for epigenetic inheritance in Arabidopsis. Molecular cell, 54(1), 30-42.

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Apoplastic Fluid Isolation from N. benthamiana

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The method of apoplastic fluid isolation from infiltrated N. benthamiana was performed according to Joosten (2012 (link)) with modifications. Briefly, two full‐size infiltrated leaves were cut off and immersed in 50 ml of cold distilled water with plant protease inhibitors (Sigma). Vacuum was applied for 2–5 min, then, after vacuum release, the leaves were surface‐dried with paper, wrapped in Parafilm sheets, and placed petal side up in a centrifuge tube. After centrifugation (1000 × g, 10 min, 4°C), around 300 μl of apoplastic fluid was gained and proteins precipitated overnight with acetone on −20°C, centrifuged and diluted in 50 µl of water. Typically, 0.1 mg/ml of protein was recovered per leaf.

Pečenková T., Pejchar P., Moravec T., Drs M., Haluška S., Šantrůček J., Potocká A., Žárský V, & Potocký M. (2022). Immunity functions of Arabidopsis pathogenesis‐related 1 are coupled but not confined to its C‐terminus processing and trafficking. Molecular Plant Pathology, 23(5), 664-678.

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Affinity Purification of Plant Protein Complexes

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Proteins were extracted from 4 grams of ∼2.5 week-old above-ground plant tissues ground to a fine powder in liquid nitrogen. The resulting powder was suspended in 14 ml lysis buffer (50 mM Tris HCl pH 7.6, 150 mM NaCl, 5mM MgCl₂, 10% glycerol, 0.5 mM Dithiothreitol (DTT), 0.1% IGEPAL, 1% plant protease inhibitors (Sigma)), filtered through two layers of Miracloth and subjected to centrifugation at 16 000 × g, 15 min, 4°C. The resulting supernatant was incubated with anti-FLAG Agarose (Sigma) for 2 h at 4°C on a rotating mixer. The agarose resin was then washed twice with lysis buffer and boiled in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer. SDS-PAGE was conducted using Tris-glycine gels and proteins were then transferred to nitrocellulose membranes for immunoblotting. Antibodies were diluted in TBST + 5% (w/v) nonfat dried milk as follows: 1:500 anti-NRP(D/E)2, 1:250 anti-RDR2, 1:500 anti-NRP(B/D/E)11 and 1:2000 anti-FLAG-HRP (Sigma). Anti-rabbit-HRP (Santa Cruz Biotechnology) diluted 1:5000 was used as secondary antibody to bind the primary antibodies. Native antibodies to NRP(D/E)2, RDR2 and NRP(B/D/E)11 were previously described (25 (link),31 (link),37 (link)).

Wendte J.M., Haag J.R., Pontes O.M., Singh J., Metcalf S, & Pikaard C.S. (2019). The Pol IV largest subunit CTD quantitatively affects siRNA levels guiding RNA-directed DNA methylation. Nucleic Acids Research, 47(17), 9024-9036.

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