Roots were sampled and frozen in liquid nitrogen. Total proteins were obtained using the Plant Total Protein Extraction Kit (Sigma) and quantified with the PerceTM660nm Protein Assay using a BSA standard curve. Membrane proteins were obtained by disrupting roots in a buffer containing a plant anti-protease cocktail (Sigma-Aldrich) and anti-phosphatases (30 mM glycerophosphate, 5 mM molybdate, and 10 mM NaF). The whole membrane fraction was then isolated by centrifugation (100000g, 4h) on a 55% sucrose cushion. Immunoblot analysis was performed on 40-50 μg of proteins using anti-GFPHRP 1:2500 (Miltenyi Biotec, 130-091-833), anti-NRT1.1 1:5000 (AS12 2611, Agrisera) and anti-PIP2.1 1:5000 61 (link). Coomassie Brilliant Blue staining of blots was used to control protein levels after electro-transfer. Un-cropped versions of the blots are provided in Supplementary Fig. 7 and 8 . Band intensity quantification was performed using a chemioluminiescent image analyzer LAS3000 (Fujifilm) and ImageGauge (Fujifilm) software.
Plant anti protease cocktail
Manufactured by Merck Group
The Plant anti-protease cocktail is a laboratory product designed to inhibit protease activity in plant samples. It is intended for use in the preparation and preservation of plant-derived samples for various analytical and experimental purposes.
Automatically generated - may contain errors
2 protocols using plant anti protease cocktail
1
Quantification of Plant Membrane Proteins
2
Quantification of Plant Membrane Proteins
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Roots were sampled and frozen in liquid nitrogen. Total proteins were obtained using the Plant Total Protein Extraction Kit (Sigma) and quantified with the PerceTM660nm Protein Assay using a BSA standard curve. Membrane proteins were obtained by disrupting roots in a buffer containing a plant anti-protease cocktail (Sigma-Aldrich) and anti-phosphatases (30 mM glycerophosphate, 5 mM molybdate, and 10 mM NaF). The whole membrane fraction was then isolated by centrifugation (100000g, 4h) on a 55% sucrose cushion. Immunoblot analysis was performed on 40-50 μg of proteins using anti-GFPHRP 1:2500 (Miltenyi Biotec, 130-091-833), anti-NRT1.1 1:5000 (AS12 2611, Agrisera) and anti-PIP2.1 1:5000 61 (link). Coomassie Brilliant Blue staining of blots was used to control protein levels after electro-transfer. Un-cropped versions of the blots are provided in Supplementary Fig. 7 and 8 . Band intensity quantification was performed using a chemioluminiescent image analyzer LAS3000 (Fujifilm) and ImageGauge (Fujifilm) software.
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