Total proteins were purified in the absence of detergent [43 (link)]. Briefly, samples containing spores were pelleted by centrifugation and rinsed with 1× PBS. Total protein concentration was measured using the bicinchoninic acid assay (BCA) using protocols from the manufacturer (Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts of protein were electrophoretically separated in 10% polyacrylamide-SDS gels using a Mini-PROTEAN Tetra cell electrophoresis unit (Bio-Rad®, Hercules, CA, USA) [40 (link)]. The proteins were transferred to 0.45 μm nitrocellulose (Bio-Rad®, Hercules, CA, USA) with a Trans-Blot Semi-Dry Transfer system (Bio-Rad®, Hercules, CA, USA). Table 1 shows the tested antibodies. Following transfer, immunoblots were viewed with enhanced chemiluminescence (ECL) substrates (Bio-Rad®, Hercules, CA, USA) using a Vilber Lourmat gel doc Fusion FX5-XT (Vilber®, Marne-la-Vallee, France). Densitometry was conducted with FUSION FX software (Vilber®, Marne-la-Vallee, France), using total protein to normalize measurements. Data represent three separate experiments from three different total protein isolations.
Mini protean tetra cell electrophoresis unit
Manufactured by Bio-Rad
Sourced in United States
The Mini-PROTEAN Tetra cell is a compact and versatile electrophoresis unit designed for the separation and analysis of proteins and nucleic acids. The unit accommodates up to four mini-gel cassettes and provides precise control over the electrophoresis process, allowing for consistent and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Gel doc xr system, by Bio-Rad (3 mentions)
Powerpac basic, by Bio-Rad (3 mentions)
Sybr gold, by Thermo Fisher Scientific (3 mentions)
Orange dna loading dye, by Thermo Fisher Scientific (2 mentions)
Ecl substrate, by Bio-Rad (1 mentions)
Trans blot semi dry transfer system, by Bio-Rad (1 mentions)
0.45 μm nitrocellulose, by Bio-Rad (1 mentions)
Lourmat gel doc fusion fx5 xt, by Vilber (1 mentions)
Fusion fx software, by Vilber (1 mentions)
Protein molecular weight marker, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Mini protean 3, by Bio-Rad (1 mentions)
1 2 bis dimethylamino ethane, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Bio-Rad (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
10 protocols using mini protean tetra cell electrophoresis unit
1
Protein Purification and Western Blotting
2
Native PAGE for DNA Analysis
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Native polyacrylamide gel (18%) was first incubated with running buffer (1 × TAE solution, pH 6.5) for 1 h at 37 °C. A volume of 30 μl of each DNA sample was mixed with 3.5 μl of glycerol, and then the mixture was added into the gel for electrophoresis. The native PAGE was carried out in a Mini-PROTEAN Tetra cell electrophoresis unit (Bio-Rad) at 37 °C, using 1 × TAE buffer at pH 6.5 and at a constant voltage of 50 V for 2 h 30 min (using Bio-Rad PowerPac Basic power supply). After 30 min of staining in 1 × SYBR gold (Invitrogen) (dissolved in a 1 × TAE buffer at pH 8.0), the gel was scanned by a Gel Doc XR+ system (Bio-Rad). In these experiments we used the following modified cargo strand containing the usual recognition domain and a 16-nt hairpin tail (bold below) that allow dye intercalation:
Cargo strand Gel: 5′-CTGCGTTTCGCAGTTT AGAAAGGAGAGA-3′
Cargo strand Gel: 5′-
3
SDS-PAGE Analysis of Purified PPO from Opuntia
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified PPO from O. ficus-indica was applied according to the Laemmli method [25 (link)]. The purified PPO of O. ficus-indica was subjected to SDS-PAGE in a Mini Protean Tetra Cell Electrophoresis Unit (Bio-Rad Laboratories, Hercules, CA, USA), with 3% stacking gel and 10% running gel. The purified PPO by affinity gel from O. ficus-indica and standard proteins were run on the SDS-PAGE gel. The purified PPO and marker were loaded in lanes, and the slab gels were 1 mm thick. The PPO and marker were run at 80 V in the stacking gel and 150 V in the separating gel (running gel). After the electrophoresis, the protein bands were visualized using Coomassie Brilliant Blue R-250. The protein molecular weight marker (Thermo Scientific, Lithuania), which was used for comparison to the molecular weight, included lysozyme (14.4 kDa), β-lactoglobulin (18.4 kDa), REase Bsp98l (25.0 kDa), lactate dehydrogenase (35.0 kDa), ovalbumin (45.0 kDa), bovine serum albumin (66.2 kDa), and β-galactosidase (116.0 kDa).
4
SDS-PAGE Analysis of Protein Profiles
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Protein profiles of WPI, YPI, YPI + WPI and YPC, prepared at 4% (w/v) at pH 8.0, were analysed using SDS-PAGE. A gradient resolving polyacrylamide gel (6%, 9%, 12%, 15%, and 18%) with 3% w/v stacking gel was cast by adapting the method of Laemmli (1970) (link). Sample buffer was prepared by using 20% glycerol, 2% SDS, 0.5% bromophenol blue (dissolved in 62.5 mM Tris-HCl buffer, pH 6.8, 1 M β-mercaptoethanol). Protein samples were mixed with the sample buffer at 1:1 (v/v), followed by heat denaturing at 90 °C for 5 min. The treated samples (20 μL) were loaded into each well and electrophoresis was conducted at 120 V for 2 h. A standard molecular weight marker (10 μL) ranging from 10 to 250 kDa was loaded into a separate well in the gel. SDS-PAGE was conducted with a BioRad Mini-PROTEAN Tetra Cell electrophoresis unit. Thereafter, gels were gently washed with Milli-Q water followed by overnight staining in Coomassie brilliant blue solution. Destaining was done with Milli-Q water under orbital shaking for about 4 h. Image scanning of gels was done with a ChemiDoc Imaging System (BioRad Inc., Canada).
5
SDS-PAGE Protein Analysis Protocol
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Electrophoresis experiments were carried out using a Mini-PROTEAN® 3 or a Mini-PROTEAN® Tetra cell electrophoresis unit (Bio-Rad Laboratories, Inc., USA). The protocols (SDS-PAGE) were carried out according to the manufacturer’s instructions. Reagents of electrophoresis grade were obtained from Bio-Rad Laboratories Inc., electrophoresis grade ammonium persulfate was purchased from Sigma Aldrich and 1,2-bis(dimethylamino)ethane was obtained from Merck Millipore. A broad range of molecular weight markers were purchased from Bio-rad Laboratories Inc. (Precision Plus Protein™ Dual Color Standards) and Thermo Scientific (Scientific PageRuler Plus Prestained Protein Ladder). Unless stated otherwise, samples were prepared following the Laemmli protocol [35 (link)]. Dithiothreitol (DTT, Bio-rad Laboratories Inc.) was selected as the reducing agent. DTT was added to a final 1x concentration of 50 mM. Samples were run on 10% SDS-PAGE vertical minigels under both non-reducing/reducing conditions (DTT included). Electrophoretic conditions were 200 V at constant current according to the manufacturer’s instructions. Minigels were stained with Coomassie blue or silver.
6
Determining Purified Enzyme Molecular Weight
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To determine the molecular weight of the purified enzyme, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was conducted according to the Laemmli method [47 (link)]. In a Mini Protean Tetra Cell Electrophoresis Unit (Bio-Rad), with 10% running gel and 3% stacking gels, purified PPO was subjected to SDS–PAGE. Purified PPO (approximately 50 μg/mL) was loaded in each lane, and the slab gels (1 mm thickness) were run at 80 volts in the stacking gel and 150 volts in the running gel. After electrophoresis, protein bands were made visible using Coomassie Brilliant Blue R-250. The molecular weight of PPO was estimated with the protein molecular weight marker (116.0, 66.2, 45.0, 35.0, 25.0, and 18.4 kDa).
7
Native PAGE Analysis of CRISPR Cleavage Activity
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Native PAGE experiments were obtained with 18% polyacrylamide (29:1 acrylamide/bisacrylamide) in TBE 10x buffer (1 M Tris, 0.9 M boric acid and 0.01 M EDTA), pH 8.3. Gel solution were prepared by mixing 5.7 ml of diH2O, 4.1 ml of TBE 10× buffer, 4.2 ml of 30% acrylamide/bisacrylamide solution, 75 μl of 10% APS, 14 μl of TEMED. A volume of 10 μl of each sample was mixed with 1 μl of 60% (w/w) Glycerol and then the mixture was added into the gel for the electrophoresis assay. O’Range Ruler 5 base pair DNA Ladder was used as the DNA standard. The samples were obtained following the same procedure reported for fluorescence trans-cleavage assay using 250 nM of FRET-based DNA reporter. The reactions were stopped at different times (0, 15, 30, 45, 60, 120 and 180 min), by heating the solutions for 10 min at 65°C in order to inactivate the Cas12a enzyme. The native PAGE was carried out in a Mini-PROTEAN Tetra cell electrophoresis unit (Bio-Rad) at room temperature, at a constant voltage of 120 V, using TBE 1× buffer (0.1 M Tris, 0.09 M boric acid and 0.001 M EDTA) at pH 8.3 for 80 min.
8
Gel Electrophoresis of DNA Samples
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Native gels (8%) were run at room temperature in running buffer (1× TBE, pH 8) at 100 V. Sample volumes of 10 μl were combined with 1 μl of 6× Orange DNA Loading Dye followed by direct loading into the gel. Denaturing polyacrylamide gels (18%) were prepared at a concentration of 7 M urea and run at room temperature in running buffer (1× TBE, pH 8) at 150 V. A sample volume of 10 μl was combined with 1 μl of Gel Loading Buffer II and 3 μl of formamide and then submitted to heat treatment at 95 °C for 5 min followed by a fast cooling in an ice-bath for 3 min. The experiments were carried out in a Mini-PROTEAN Tetra cell electrophoresis unit (Bio-Rad) using Bio-Rad PowerPac Basic power supply. If appropriate, gels were first scanned by a ChemiDoc MP imaging system (Bio-Rad) to detect fluorophores (FAM and CFR610), then stained with SYBR Gold and imaged. Reaction yields were estimated based on band intensities by densitometry using Image Lab software from Bio-Rad (version 6.0.1).
9
Native PAGE for Monitoring TBP-Induced DNA Dynamics
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A native polyacrylamide gel (10%) was used to monitor the binding dynamics of the TBPinduced strand displacement network. An aliquot of 10 µl of each sample (see Figure SI3) was mixed with 2uL of 6x Orange DNA Loading Dye (Thermo Fischer Scientific) and loaded into the gel. The native PAGE was performed in a Mini-PROTEAN Tetra cell electrophoresis unit (Bio-Rad) at room temperature using 1 × TBE buffer at pH 8.3 and at a constant voltage of 90 V for 1 h (using Bio-Rad PowerPac Basic power supply). To visualize DNA bands, a 30 min staining with 1 × SYBR gold (Invitrogen) in 1 × TBE buffer was applied. The gel was imaged under a Gel Doc XR+ system (Bio-Rad). To visualize TBP, the gel was subsequently stained with InstantBlue® Protein Gel Stain, according to the manufacturer's instruction. Briefly, 40 mL of staining solution were added to the gel, which was imaged under the Gel Doc XR+ system once the bands had become visible at naked eye.
10
Native PAGE for Monitoring TBP-Induced DNA Dynamics
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A native polyacrylamide gel (10%) was used to monitor the binding dynamics of the TBPinduced strand displacement network. An aliquot of 10 µl of each sample (see Figure SI3) was mixed with 2uL of 6x Orange DNA Loading Dye (Thermo Fischer Scientific) and loaded into the gel. The native PAGE was performed in a Mini-PROTEAN Tetra cell electrophoresis unit (Bio-Rad) at room temperature using 1 × TBE buffer at pH 8.3 and at a constant voltage of 90 V for 1 h (using Bio-Rad PowerPac Basic power supply). To visualize DNA bands, a 30 min staining with 1 × SYBR gold (Invitrogen) in 1 × TBE buffer was applied. The gel was imaged under a Gel Doc XR+ system (Bio-Rad). To visualize TBP, the gel was subsequently stained with InstantBlue® Protein Gel Stain, according to the manufacturer's instruction. Briefly, 40 mL of staining solution were added to the gel, which was imaged under the Gel Doc XR+ system once the bands had become visible at naked eye.
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