We tested several cell wall digestion enzymes from multiple manufacturers and found the performance of the enzymes varied between manufacturers, as well as between batches from the same manufacturers. We tested driselase (Sigma), cellulase (Sigma), cellulase R10 (Duchefa, Yakult), cellulase RS (Duchefa, Yakult), pectolyase (Duchefa; discontinued), pectinase (Sigma), macerozyme R10 (Duchefa) and hemicellulase (Sigma) for conventional whole-mount protocol. We chose cellulase RS (Duchefa), hemicellulase (Sigma) and pectinase (Sigma) or macerozyme R10 (Duchefa) for expansion microscopy based on their performance and availability.
Pectinase
Manufactured by Merck Group
Sourced in United States, Germany, Japan, France
Pectinase is an enzyme used in laboratory applications. It functions to break down pectin, a complex carbohydrate found in plant cell walls.
Automatically generated - may contain errors
Lab products found in correlation
Cellulase, by Merck Group (30 mentions)
Onozuka r 10, by Serva Electrophoresis (18 mentions)
Pectolyase, by Merck Group (9 mentions)
Driselase, by Merck Group (5 mentions)
Cellulase onozuka r 10, by Serva Electrophoresis (5 mentions)
Hemicellulase, by Merck Group (4 mentions)
Pectin, by Merck Group (4 mentions)
Aspergillus niger, by Merck Group (3 mentions)
Xylanase, by Merck Group (3 mentions)
α amylase, by Merck Group (3 mentions)
Macerozyme, by Serva Electrophoresis (3 mentions)
Dmrb microscope, by Leica (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (3 mentions)
Gallic acid, by Merck Group (2 mentions)
α glucosidase, by Merck Group (2 mentions)
Acarbose, by Merck Group (2 mentions)
Pectolyase, by Duchefa Biochemie (2 mentions)
Cellulase, by Duchefa Biochemie (2 mentions)
Q500mc image analyser, by Leica (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
123 protocols using pectinase
1
Optimizing Cell Wall Enzymatic Digestion
2
Preparation and analysis of meiotic and mitotic chromosomes
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Preparation of meiotic chromosome squashes followed published methodology [35] (link), [37] (link). Briefly, individual anthers were isolated using fine needles in a watch glass with a 10 mM citrate buffer then digested enzymatically for 2 h at 37°C in a mixture comprising 10% pectinase (Sigma, cat. no. P-0690), 0.65%, cellulase “Onozuka R-10” (Serva, cat. no. 16419.02), 0.5% cellulase (Calbiochem, cat. no. 21 947), 0.15% cytohelicase (Sigma, cat. no. C-8274) and 0.15% pectolyase (Sigma, cat. no. P-3026). The anthers were transferred to a slide in a drop of 45% acetic acid, covered with a coverslip, gently squashed and frozen on dry ice. The coverslips were flicked off with a blade and the slides were air-dried.
For B. pinnatum PI 345982, mitotic chromosome preparations were made using the methodology described in [37] (link), [38] (link). In brief, enzymatic digestion of roots was carried out for 2 h at 37°C in a mixture comprising 20% pectinase (Sigma, cat. no. P-0690), 1% cellulase (Calbiochem, cat. no. 21 947) and 1% cellulase “Onozuka R-10” (Serva, cat. no. 16419.02) in 10 mM citrate buffer. The meristems were extruded in 45% acetic acid and transferred to a slide, covered with a coverslip and squashed. Further steps in the procedure were same as for meiotic chromosome preparations.
For B. pinnatum PI 345982, mitotic chromosome preparations were made using the methodology described in [37] (link), [38] (link). In brief, enzymatic digestion of roots was carried out for 2 h at 37°C in a mixture comprising 20% pectinase (Sigma, cat. no. P-0690), 1% cellulase (Calbiochem, cat. no. 21 947) and 1% cellulase “Onozuka R-10” (Serva, cat. no. 16419.02) in 10 mM citrate buffer. The meristems were extruded in 45% acetic acid and transferred to a slide, covered with a coverslip and squashed. Further steps in the procedure were same as for meiotic chromosome preparations.
3
Pectinase Treatment and Callose Visualization
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For exogenous pectinase treatment, Arabidopsis seedlings grown for 4 days after transferring on normal or P-deficient ½ MS medium plates were collected, and 7-day-old seedlings were incubated with liquid ½ MS medium containing 25 U/ml pectinase (Sigma-Aldrich, Cat#P2611) for 20 min at 30°C. Then, seedlings were washed by liquid ½ MS medium and photographed under an epifluorescence microscope (Leica DM4B).
To detect callose deposition in roots, aniline blue staining was performed according to a previously described method (Muller et al., 2015 (link)). Briefly, 7-day-old seedlings was stained with 0.1% (w/v) aniline blue (Sigma-Aldrich, Cat#415049) in 100 mm phosphate buffer (pH 7.2) for 1.5 h. Root samples were placed in 20% (v/v) glycerol to observe under a spinning-disk confocal microscope with a 405-nm excitation laser and 445/50-nm emission filter with a 20x objective (Zeiss, Observer SD).
To detect callose deposition in roots, aniline blue staining was performed according to a previously described method (Muller et al., 2015 (link)). Briefly, 7-day-old seedlings was stained with 0.1% (w/v) aniline blue (Sigma-Aldrich, Cat#415049) in 100 mm phosphate buffer (pH 7.2) for 1.5 h. Root samples were placed in 20% (v/v) glycerol to observe under a spinning-disk confocal microscope with a 405-nm excitation laser and 445/50-nm emission filter with a 20x objective (Zeiss, Observer SD).
4
Cytogenetic Analysis of Plant Root Tips
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Plants were cultivated in plastic pots filled with perlite in the greenhouse. Root tips were pre-treated in ice cold water for 24–33 h and fixed in fresh ethanol-acetic acid fixative (3:1, v/v).
The fixed root tips were washed in distilled water (twice) and citrate buffer (0.01 M citric acid and 0.01 M sodium citrate buffer, pH 4.8) for 5 min each. Thereafter, the root tips were treated in an enzyme mixture [1% (w/v) pectinase, 1% (w/v) pectolyase and 20% (v/v) pectinase (Sigma, St. Louis, MO, USA) in citrate buffer] for 3–4 h at 37 °C. After treatment, the digested tissue was washed in distilled water.
The slides were prepared using the smear method [48 (link)] according to a previous report [49 (link)] with several modifications. The digested tissue was carefully transferred to a microscope slide, and a suspension was produced with needles. Cold 75% acetic acid was then immediately added to the suspension, after which the slide was placed on a hot plate and stirred with a needle to spread the cells. Finally, 150 μl of cold ethanol-acetic acid fixative was added, and the slides were washed with ethanol and air-dried.
The fixed root tips were washed in distilled water (twice) and citrate buffer (0.01 M citric acid and 0.01 M sodium citrate buffer, pH 4.8) for 5 min each. Thereafter, the root tips were treated in an enzyme mixture [1% (w/v) pectinase, 1% (w/v) pectolyase and 20% (v/v) pectinase (Sigma, St. Louis, MO, USA) in citrate buffer] for 3–4 h at 37 °C. After treatment, the digested tissue was washed in distilled water.
The slides were prepared using the smear method [48 (link)] according to a previous report [49 (link)] with several modifications. The digested tissue was carefully transferred to a microscope slide, and a suspension was produced with needles. Cold 75% acetic acid was then immediately added to the suspension, after which the slide was placed on a hot plate and stirred with a needle to spread the cells. Finally, 150 μl of cold ethanol-acetic acid fixative was added, and the slides were washed with ethanol and air-dried.
5
Antioxidant Extraction and Quantification
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The extraction solvents consisted of variable percentages of absolute ethanol EssentQ® (Scharlab, Barcelona, Spain) diluted in different citrate/phosphate buffers at pH 4.0, 5.0 and 6.0 obtained by solving calcium citrate (Panreac S.L.U., Castellar del Vallés, Spain) and sodium dihydrogenphosphate (Panreac S.L.U., Castellar del Vallés, Spain) in Milli-Q water obtained by filtration through a MilliPore system (Bedford, MA, USA). The enzyme employed for the extraction was a pectinase obtained from Aspergillus niger (Merck KgaA, Darmstadt, Germany).
For the determination of the antioxidant capacity of the extract, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Trolox (Merck KgaA, Darmstadt, Germany) were used.
In order to quantify the amount of phenolic compounds present in the extract, gallic acid, Folin–Ciocalteu reagent and sodium carbonate (Merck KgaA, Darmstadt, Germany) were employed.
Two solvents were employed to separate the constituents of the extracts as follows: HPLC-grade acetonitrile (Panreac S.L.U, Castellar del Vallés, Spain) and Milli-Q water, both acidified using 2% glacial acetic acid (Panreac S.L.U, Castellar del Vallés, Spain). Quercetin 3-O-glucoside (Q3GLU) (Merck KgaA, Darmstadt, Germany), were used as standard for the quantification.
For the determination of the antioxidant capacity of the extract, 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Trolox (Merck KgaA, Darmstadt, Germany) were used.
In order to quantify the amount of phenolic compounds present in the extract, gallic acid, Folin–Ciocalteu reagent and sodium carbonate (Merck KgaA, Darmstadt, Germany) were employed.
Two solvents were employed to separate the constituents of the extracts as follows: HPLC-grade acetonitrile (Panreac S.L.U, Castellar del Vallés, Spain) and Milli-Q water, both acidified using 2% glacial acetic acid (Panreac S.L.U, Castellar del Vallés, Spain). Quercetin 3-O-glucoside (Q3GLU) (Merck KgaA, Darmstadt, Germany), were used as standard for the quantification.
6
Casein Microparticle Preparation via Pectin Depletion
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The following working solutions were prepared for the individual preparation steps. A pectinase solution with an enzyme activity of 36 units/mL was prepared by adding 0.47 mL of pectinase from Aspergillus niger (Merck, Darmstadt, Germany) to 10 g of SMUF buffer for film hydrolysis. A casein dispersion (10% w/w) was prepared by adding 5 g casein powder to 45 g SMUF solution. The dispersion was stirred at room temperature for one hour, then at 4 °C for 4 h, and finally at 37 °C for another hour, in each case at 200 rpm. The pH of the casein dispersion was 6.7.
Casein microparticles were prepared by pectin-induced depletion flocculation of casein micelles and subsequent solidification of the resulting aggregates by film drying. Aggregates were initially formed by mixing a 0.3% micellar casein dispersion and a 3% pectin solution at pH 6.7 and room temperature. A total of 3.9 g of the aggregate solution was added to a glass petri dish and dried for 16 h under controlled laboratory conditions (T = 22 °C, RH 45%). The solidified casein micro-particles were then released from the film matrix by enzymatic hydrolysis with pectinase and separated from pectin residues by centrifugation at 1500 RCF for 10 min.
Casein microparticles were prepared by pectin-induced depletion flocculation of casein micelles and subsequent solidification of the resulting aggregates by film drying. Aggregates were initially formed by mixing a 0.3% micellar casein dispersion and a 3% pectin solution at pH 6.7 and room temperature. A total of 3.9 g of the aggregate solution was added to a glass petri dish and dried for 16 h under controlled laboratory conditions (T = 22 °C, RH 45%). The solidified casein micro-particles were then released from the film matrix by enzymatic hydrolysis with pectinase and separated from pectin residues by centrifugation at 1500 RCF for 10 min.
7
Xylan Epitope Detection by Pectin Pretreatment
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The detection process of masking xylans epitope LM27 by pectins was carried out according to Davies et al. [44 (link)] with modifications. The sections on the glass slides were first pre-treated with 2% pectinase (Merck KGaA, Darmstadt, Germany, formerly Sigma, St. Louis, MO, USA) in PBS buffer for 30 min, washed 3 times with PBS buffer. Then, the detection process of xylans with use of LM27 antibody, was carried out as described above. Some sections were carried out with control reaction using LM19 antibody.
8
Tobacco Pollen Tube Protoplast Isolation
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Tobacco pollen tube protoplasts were prepared as follows (Yu et al., 2006 (link)). The collected pollen grains were first germinated in GM [1.0mM KNO3, 1.0mM Ca (NO3)2, 1.0mM MgSO4, 1.0mM H3BO3, and 20% sucrose, pH 5.8] at 25 °C. When the pollen tubes had emerged and were vigorously growing, they were transferred into an enzyme solution containing 1% cellulose (Onozuka R-10) and 1% pectinase (Merck) dissolved in bath solution, and incubated at 28 °C for 1h to release pollen protoplasts.
9
Cytophotometric Analysis of Plant Nuclei
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For cytophotometric analysis, root tips (3–4 mm) of control and transgenic plants were fixed in a mixture of ethanol and acetic acid (3:1 ratio) for 3 h. The root meristem zone (up to 1–2 mm) was separated and placed for 2 h at 37 °C in a macerating mixture containing 0.4% cellulase (Sigma-Aldrich Co., St. Louis, MO, USA) and 0.4% pectinase (Merck, Darmstadt, Germany), then stained using the Feulgen method (Schiff reagent, Merck) for 2 h (hydrolysis time in 5N HC1 at 22 °C, 40 min) and permanent preparations were prepared. To determine the DNA content, the nuclei of meristematic cells were measured using an SMP-20 Opton cytophotometer (Carl Zeiss, Obercochen, Germany) with a 16 × objective, 10 × eyepiece, and 0.16 mm probe. The nuclei of meristematic root cells at the telophase (2C) or metaphase (4C) stages were used as a standard. The sample for each experimental point was at least 300 nuclei from cells of 10–12 roots. The data were analyzed in the Statistics 6.0 program.
10
Cytogenetic Analysis of Plant Root Tips
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For cytogenetic analysis, seeds were germinated and root tips were collected and pretreated with 8-hydroxyquinoline for 20 h at 10°C, fixed in ethanol:acetic acid (3:1; v/v) from 2 to 24 h at room temperature and stored at –20°C. The fixed roots were washed in distilled water and digested in 2% cellulase (Onozuka) and 20% pectinase (Merck) at 37°C for 90 min. Then apical meristems were squashed in 45% acetic acid under a coverslip. The coverslip was removed in liquid nitrogen.
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