The samples of leaf were immediately harvested after a plant being uprooted, weighed and frozen in liquid nitrogen followed by grinding in chilled pestle and mortar to a fine powder. This powder was extracted with 40 mM (w/v) Tris-HCl, pH 7.5, 2 mM (w/v) EDTA, 0.07% (w/v) β-mercaptoethanol, 2% (w/v) PVP and 1% (v/v) Triton X-100. The extract was centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was mixed with 6-X protein-dye containing 240 mM Tris-HCl (pH 6.8), 40% glycerol, 8% SDS, 0.04% bromophenol blue and 5% beta-mercaptoethanol. The samples containing 40 μg proteins were loaded on 12.5% polyacrylamide gel on PROTEAN II (Bio-Rad, Hercules, CA, USA). The protein concentration was determined by Bradford method using BSA (bovine serum albumin) as a standard curve. After electrophoresis, the gels were stained with a commercial available silver stain according to manufacturer’s instructions (Bio-Rad, Hercules, CA, USA).
Protean 2
Manufactured by Bio-Rad
Sourced in United States
The Protean II is a vertical gel electrophoresis system designed for high-resolution protein separation. It features a modular design and is compatible with a wide range of gel sizes and formats, allowing for flexibility in experimental setup.
Automatically generated - may contain errors
Lab products found in correlation
Silver stain, by Bio-Rad (2 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
Broad range molecular weight standard, by Bio-Rad (1 mentions)
Mini protean 2 system, by Bio-Rad (1 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (1 mentions)
Protean ief cell, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Ts primer, by Merck Group (1 mentions)
Chemiluminescent detection system, by Thermo Fisher Scientific (1 mentions)
Anti caspase 3, by Thermo Fisher Scientific (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti bim, by GeneTex (1 mentions)
Anti bcl 2, by GeneTex (1 mentions)
Anti bax, by GeneTex (1 mentions)
Typhoon 9200 phosphor imager, by Cytiva (1 mentions)
Coomassie brilliant blue g 250, by Merck Group (1 mentions)
High resolution scanner, by Epson (1 mentions)
14 protocols using protean 2
1
Leaf Protein Extraction and SDS-PAGE
2
SDS-PAGE Protein Separation Protocol
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Protein separation by SDS-PAGE was performed following the method described by Laemmli37 (link) using a Mini Protean II system (Bio-Rad, 2000, CA, USA). Samples were mixed with 4 × sample buffer containing 50 mM Tris–HCl pH 6.8, 20% glycerol, 10% SDS, 10% β-mercaptoethanol and 0.05% bromophenol blue. The samples were then boiled for 10 min and loaded into either 10% or 15% polyacrylamide gel. A broad-range molecular weight standard (Bio-Rad, 1610317, CA, USA) was also loaded into the gel. Electrophoresis was carried out at 90-V and room temperature using a vertical electrophoresis unit (Bio-Rad Protean II, CA, USA). After electrophoresis, the gels were stained with Coomassie blue solution (0.05% (w/v) Coomassie Brilliant Blue R-250, 7% v/v acetic acid, 40% v/v methanol) for 2 h. Protein bands were then visualized by soaking the gels in a distaining solution (7% v/v acetic acid and 40% v/v methanol) for 2 h. Gel images were digitalized using a gel imager (Chemi Doc XRS, Bio-Rad, CA, USA). The relative molecular mass of protein bands was determined by comparing their migration with the molecular weight standard.
3
Protein separation by 2D-PAGE
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Immediately before, the isoelectric focusing sample was diluted to 200 µL in the rehydration buffer (up to a concentration of 0.35 mg/mL protein). The samples were loaded onto IPG strips (4–7 pH, 11 cm length) and the process was carried out in the Protean IEF cell (active rehydration at 50 V, 12 h, focusing conditions: 8000 V up to 35,000 V-h, a maximum current of 50 µA/IPG strip) (Bio-Rad, Warszawa, Poland).
Before loading onto SDS-polyacrylamide gels, IPG strips were incubated for 15 min in an equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS) containing 5 mM DTT and then for another 15 min in equilibration buffer containing 15 mM iodoacetamide. The SDS-polyacrylamide gels (20 × 20 cm, 1.5 mm, T = 11%, C = 2.6%) were proceeded in accordance with Laemmli (1970) [23 (link)]. The second dimension was performed using a Protean II according to the manufacturer’s instructions (Bio-Rad, Warsaw, Poland).
Before loading onto SDS-polyacrylamide gels, IPG strips were incubated for 15 min in an equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 30% glycerol, 2% SDS) containing 5 mM DTT and then for another 15 min in equilibration buffer containing 15 mM iodoacetamide. The SDS-polyacrylamide gels (20 × 20 cm, 1.5 mm, T = 11%, C = 2.6%) were proceeded in accordance with Laemmli (1970) [23 (link)]. The second dimension was performed using a Protean II according to the manufacturer’s instructions (Bio-Rad, Warsaw, Poland).
4
Plant Protein Extraction and Characterization
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The plant tissue (1g) was extracted using freshly prepared protein extraction buffer as previously described by Muneer et al., 2016 (link). The extraction buffer constituted (pH 7.5) 40 mM (w/v) Tris-HCl (pH 7.5), 2 mM (w/v) EDTA, 0.07% (w/v) β-mercaptoethanol, 2% (w/v) PVP and 1% (v/v) Triton X-100. The extract was centrifuged at 13,000 rpm for 10 min at 4°C. The supernatant was mixed with protein-dye and 20 µg proteins were loaded on 12.5% polyacrylamide gel on PROTEAN II (Bio-Rad, Hercules, CA, USA). The protein concentration was determined by the Bradford method using BSA (bovine serum albumin) as a standard curve. Following electrophoresis, the gel was stained with commercially available Coomassie brilliant blue stain (CBBS) according to manufacturer’s instruction (Bio-Rad).
5
SDS-PAGE Analysis of Defatted Milk Proteins
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The defatted milk sample was dissolved in buffer (50 mM Tris–HCl pH 6.8, 2% SDS, 10% glycerol, 1% β-mercaptoethanol, 12.5 mM EDTA and 0.02% bromophenol blue). Samples were heated at 95°C for 5 min and the supernatant was used in electrophoresis. Polyacrylamide gel electrophoresis (PAGE) was carried out in a vertical electrophoresis apparatus (Protean II, Bio-Rad, Richmond, CA, United States) at 75 V for 4 h in a cold chamber. The separation gel was stained with Coomassie blue for 12 h and destained for the same time. The gel image was densitogrammed using FluorChem software to quantify the percentage of each band with respect to the total proteins.
6
Protein Extraction from Rose Cultivars
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Protein extraction was done according to our previous methods [27 (link)]. The frozen powdered samples from all cut rose cultivars was extracted with extraction buffer (pH 7.5) containing 40 mM (w/v) Tris-HCl, pH 7.5, 2 mM (w/v) EDTA, 0.07% (w/v) β-mercaptoethanol, 2% (w/v) PVP and 1% (v/v) Triton X-100. The extract was centrifuged at 13,000 rpm for 10 min at 4 °C. The supernatant was mixed with protein-dye and 20 μg proteins were loaded on 12.5% polyacrylamide gel on PROTEAN II (Bio-Rad, Hercules, CA, USA). The protein concentration was determined by the Bradford method using BSA (bovine serum albumin) as a standard curve. After electrophoresis, the gels were stained with a commercial available silver stain according to manufacturer’s instructions (Bio-Rad, Hercules, CA, USA).
7
Telomerase Activity Quantification in Ovarian Samples
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Ovarian samples were homogenized using RNase-free pestle and mortar and lysed as in [74 (link)]. The CY5-labelled telomerase-substrate primer (TS-primer: 5′-Cy5-AATCCGTCGAGCAGAGTT-3′, Sigma-Aldrich [75 (link)]); was elongated, and elongation products were amplified together with an internal control, as in [75 (link)]. Two protein concentrations were used for each sample (0.5 and 2.5 μg). A negative control was included by preincubating each sample extract with RNase (Roche Diagnosis) for 10 min at 30°C as in [76 (link)]. Jurkat cells were used as a positive control. Electrophoresis was run in an acrylamide: bisacrylamide 19:1 gel (Bio-Rad) using Protean II (Bio-Rad) electrophoresis chambers. Gels with Cy5 signals were imaged wet in ChemiDoc (Bio-Rad). Image Lab software (version 5.0) was used for quantification of the TRAP image shown in Figure 2 .
8
Western Blot Analysis of Apoptosis Proteins
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Protein extraction was carried out from approximately 6 million cells treated for 6, 16, 24 and 48 h with the MTAs at the corresponding IC50 using a lysis buffer (Tris HCl pH 8.0 20 mM, NaCl 137 mM, Glycerol 10%, Np40 1%, EDTA 10 mM) [34] , and the protein concentration was determined by Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce). Fifteen μg of protein were subjected to an 11% SDS-PAGE gel electrophoresis using a mini-gel system (Protean II; Bio-Rad Laboratories). Proteins were blotted using polyvinylidene fluoride (PVDF) membranes [35] , [36] . The transferred membranes were blocked in 5% (w/v) BSA/TTBS followed by overnight incubation with the following primary antibodies: anti-α-Tubulin (Sigma–Aldrich); anti-Bim; anti-Bax; anti-Bcl-2 (Gene-Tex); anti-procaspase-3 (Novus) or anti-caspase-3 (Thermo-Fisher Scientific). Next, the membranes were incubated with the correspondent secondary antibody, either anti-mouse IgG (Sigma–Aldrich) or anti-rabbit IgG (Cell Signaling), for 1 h at room temperature. The bands were visualized using a Chemiluminescent Detection System (Thermo-Fisher Scientific) [22] . Image J software was used to quantify and compare the density of bands [37] (link).
9
SDS-PAGE Analysis of P. sterna Shell Proteins
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Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli [34 (link)]. Samples ASM (30 μg of protein) and AIM (64 μg of protein) of the shell from P. sterna were mixed with sample buffer 4× (0.5 M Tris-HCl pH 6.8, 20% glycerol, 10% SDS, 10% β-mercaptoethanol and 0.05% bromophenol blue) and boiled for 10 min, then loaded into a 16% polyacrylamide gel. Broad range molecular weight standard (Bio-Rad 1610317, California, USA) was loaded into the gel. Electrophoresis was conducted at 90-V at room temperature, using a vertical electrophoresis unit (Bio-Rad Protean II, California, USA). After electrophoresis, the gel was stained with Coomassie Brilliant Blue R250 (CBB) for 2 h, washed out and analyzed for protein, using a gel imager (Chemi Doc XRS, Bio-Rad, California, USA). Also, proteins were stained with silver nitrate [35 (link),36 (link)]. The same procedure was followed for purified protein from P. sterna (Ps19), loading 6.8 μg of protein.
10
Oligomer Size Separation by Vertical PAGE
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A vertical polyacrylamide gel electrophoresis setup (Bio-Rad Protean II) is used for oligomers size separation. An amount of 5 μg of previously dried DNA are rehydrated with 10 μl of formamide and 3 μl of gel loading solution (30% glycerol, 0.025% bromophenol blue, 0.25% xylene cyanol and water), and loaded in denaturing polyacrylamide gel (0.75–1.5 mm thick, 7 M urea, TBE buffer and acrylamide:bisacrylamide 37.5:1), and run for 1–4 h at 30 V cm−1 voltage. Gels are stained for 20 min in 0.5 μg ml−1 EtBr TBE buffer, viewed on ultraviolet–visible transilluminator and images are acquired with Typhoon-9200 Phosphor Imager (Amersham Pharmacia Biotech). The ladder is the same for each gel and it is composed of DNA oligomers synthesized with repetitions of the D1p 12-mer sequence (12-mer 5′- CGCGAATTCGCG -3′, 24-mer 5′- CGCGAATTCGCGC - GCGAATTCGCG -3′, 36-mer 5′- CGCGAATTCGCG - CGCGAATTCGCG - CGCGAATTCGCG -3′ and 48-mer 5′- CGCGAATTCGCG - CGCGAATTCGCG - CGCGAATTCGCG - CGCGAATTCGCG -3′ from Biomers). Details in Supplementary Methods .
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