A bacterial pellet aliquot, after thawing, was dissolved in a resuspension buffer (20 mM Hepes/Tris, 300 mM NaCl pH 7.5) containing protease inhibitor cocktail according to manufacturer instructions. The volume of resuspension buffer was calculated proportionally to the OD measured at 600 nm wavelength: about 20 mL for a pellet deriving from 400 mL of cell culture. The bacterial suspensions were sonified in pulses, in an ice bath, for 5 min (1 s on, and 1 s off) using a Branson SFX 550 sonifier (Emerson, Round Rock, TX, USA) at 30%. The cell lysate was centrifuged at 13,000× g and the pellet obtained was stored at −20 °C or further analyzed. Protein concentration was measured by the Lowry method, modified for the presence of detergents [37 (link)]. Proteins were separated by 12% (w/v) SDS–PAGE according to Laemmli [38 (link)] using the Hoefer SE260 mini-vertical unit and stained with Coomassie Brilliant Blue. Quantitative evaluation of Coomassie-stained protein bands was carried out using the Chemidoc imaging system equipped with Image Lab 6.1 software (Bio-Rad, Hercules, CA, USA).
Se260
Manufactured by Hoefer
Sourced in United States
The SE260 is a piece of laboratory equipment designed for sample preparation and analysis. It is a centrifugal concentrator that can be used to evaporate solvents and concentrate samples. The SE260 operates at a range of speeds and can accommodate various sample sizes.
Automatically generated - may contain errors
Lab products found in correlation
Image lab 6, by Bio-Rad (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Trans blot turbo rta transfer kit, by Bio-Rad (1 mentions)
Rabbit anti mouse gapdh, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg h l hrp, by Bio-Rad (1 mentions)
Coomassie brilliant blue, by Thermo Fisher Scientific (1 mentions)
Acetic acid, by Merck Group (1 mentions)
Bromophenol blue, by Merck Group (1 mentions)
1d image analysis software, by Kodak (1 mentions)
Dc290, by Kodak (1 mentions)
0.45 m nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Pierce ecl western blot substrate, by Thermo Fisher Scientific (1 mentions)
12 protocols using se260
1
Bacterial Protein Extraction and Quantification
2
SDS-PAGE Protein Analysis Protocol
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A volume of the sample containing 20 μg of protein was mixed with a sample buffer (5% β-mercaptoethanol; tris-HCl, 0.02 mol L−1; pH, 6.8; 4% SDS; 27% glycerol; and 0.1% bromophenol blue). Then, the mixture solution and the low molecular weight marker (LMW) (14 to 97 kDa; GE Healthcare, Illinois, USA) were denatured separately at 95°C for 5 min. After this, they were both applied on the top of the 12.5% polyacrylamide gel (8 × 10 cm) in a vertical electrophoresis system (SE 260-Hoefer, San Francisco, California, USA), and LMW was inserted in the left column. The electrophoresis system was connected to a power supply in which the following running program was established: 150 V at 50 mA for 1.5 hours. After the run, the gel was placed in fixation buffer (40% alcohol and 10% acetic acid) for 1 hour, subsequently replaced by a Coomassie blue dye solution (8% sulfate ammonium, 0.8% phosphoric acid, 0.08% Coomassie Brilliant Blue G-250, and 20% methanol) for 24 hours, under agitation. Finally, the gel was conditioned with distilled water under stirring for five days to remove excess dye. After all the mentioned processes, it was possible to observe the total protein band profiles in the gel.
3
Protein Separation by Electrophoresis
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All procedures should be performed at room temperature unless noted otherwise. The authors use a Hoefer SE 260 electrophoresis system, but the protocol can be adapted to any other electrophoresis system (see Note 9).
4
Aortic wall IL-1β and CD68 analysis
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To evaluate the levels of IL-1β and CD68 in the aortic wall of the Ang-II + 01BSUR group and Ang-II group, Western immunoblot analysis was performed. Aortic tissues (n = 3 per group) were homogenized and solubilized in RIPA buffer (pH = 7.4). The protein concentration was determined using UV-spectrometry at 280 nm (Evolution 220 UV-Visible Spectrometer, Thermo ScientificTM). 5 mg/ml total protein per lane diluted in SDS loading buffer was loaded onto 4-12% Tris-Glycine Gels (ServaGel TG Prime Vertical Tris-Glycine Gel 4-12%). Proteins were separated by SDS-PAGE (Hoefer SE260). To assure equal amounts of total protein loaded per lane, GAPDH was used as loading control. Due to the identical molecular weights of the targeted proteins CD68 and GAPDH, 2 gels were run simultaneously. Proteins were transferred to PVDF membranes using Trans-Blot Turbo RTA transfer kit (Bio-Rad). Primary antibodies (Rabbit anti-Mouse IL-1β, Bio-Rad 1:500; Rabbit anti-Mouse CD68, antibodies.com , 1:500; Rabbit anti-Mouse GAPDH, Invitrogen 1:1000) were detected using a secondary HRP-labeled antibody (Goat anti-Rabbit IgG (H/L): HRP, Bio-Rad 1:1000) and SeramunBlau® blotting substrate (Seramun Diagnostica GmbH). Protein bands were quantified by ImageJ (Version 1.51).
5
Polyacrylamide Gel Electrophoresis of bDNA
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Polyacrylamide gel electrophoresis of all self assembled bDNA samples were carried out in mini vertical gel electrophoresis system (Hoefer, SE260). 10% polyacrylamide gel was prepared by adding 7.5 ml of 30% acrylamide solution (acrylamide: bis-acrylamide :: 29:1), 450 μl of 50 X TAE buffer (Tris 2 M, pH 8.0, acetic acid 1 M, EDTA 100 mM), 2.81 ml of 100 mM Mg (Ac)2, 300 μl of 10% APS, 15 μl of TEMED and the volume was made up to 22.45 ml with MilliQ water (18.2 MΩ.cm). This is sufficient for making two numbers of gel slab of dimension 10 × 10 cm. Electrophoresis for all samples was conducted at 150 V and 4 °C for an appropriate time according to the nature of the sample. After electrophoresis the gels were stained in EtBr solution (0.5 μg/ml) for 1 h and then viewed and documented in gel documentation system (FluorChem E, Cell Bioscience).
6
SDS-PAGE Analysis of Protein Samples
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Using SDS-PAGE, hydrolyzed samples from various treatments were analyzed [36 ,37 (link)]. The samples (containing 40 µg protein) were mixed 1:1 with loading buffers that contained 0.125 M Tris-HCl, 4% SDS, 20% v/v glycerol, 0.2 M DTT, and 0.02% bromophenol blue (Merck) at a pH of 6.8. This diluted sample was preheated for 5 min in a boiling water bath at 100°C. The analysis was performed in a vertical electrophoresis unit (SE 260, Hoefer, San Francisco, CA) with 1.5 mm (17%) polyacrylamide gel slabs to separate the protein, at a constant current of 30 mA per gel. The separated protein bands were stained with a solution containing 7% acetic acid(Merck), 0.5% Coomassie Brilliant Blue (Thermo Fisher Scientific) R-250, and 40% methanol (Germany). This excess stain was removed with a solution containing 40% methanol and 7% acetic acid, with the gel was recorded with an electronic scanner (Umax Power Look 2100, UMAX Technologies, Fremont, CA, USA).
7
Fluorescent Carbohydrate Separation by PAGE
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Carbohydrates labelled with ANTS, APTS or ANDS were separated and analysed with polyacrylamide gels according to the published methodology [31] (link). For electrophoretic separation, a water-cooled electrophoresis system SE260 (Hoefer) was used. The gels were hand-cast between Mini-PROTEAN glass plates (Bio-Rad Ltd.). Typical gels were 8 × 7 cm and 1.0 or 1.5 mm thick. Fluorescently-labelled carbohydrate samples were mixed with glycerol prior to loading on the gel. The separation was carried out at constant 300 V and the run was monitored under UV-light to ensure efficient separation of individual bands. Separation gel: 20% (w/v) polyacrylamide gel and 0.5% (w/v) N,N′-methylenebisacrylamide in 0.1 M Tris adjusted to pH 8.2 with boric acid. Stacking gel: 8% (w/v) polyacrylamide gel and 0.2% (w/v) N,N′-methylenebisacrylamide in 0.1 M Tris adjusted to pH 8.2 with boric acid. Separation buffer: 0.1 M Tris adjusted to pH 8.2 with boric acid. After separation, the gel cassettes were removed from the electrophoresis chamber and visualised with a Fuji FLA-7000 imaging system equipped with the filter set for CY2 detection (473 nm excitation, 530 nm emission filter, Fuji Photo Film Co. Ltd).
8
Extraction and Analysis of Oat Acid Phosphatases
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Root and shoot tissues of four oat cultivars (1 g) were ground in a mortar chilled with liquid N2, then 4 ml of extraction buffer was added (100 mM Na-acetate, pH 5.0, 2 mM EDTA, 20 mM CaCl2, 5 mM DTT and 60 mg PVPP), the solution was gently mixed at 4 °C for 60 min and subsequently centrifuged at 10,000 rpm, then pellet was discarded. Equal protein amounts (10 µg for individual shoots per lane and 6 µg for roots) were loaded onto a discontinuous native PAGE (5% (w/v) stacking gel, 10% (w/v) resolving gel). The native gels were run at 4 °C using a mini-gel system (SE 260; Hoefer, Holliston, MA, USA; Amersham, Little Chalfont, UK) and washed in 0.1 mM Na-acetate buffer. Approximate masses of APase isoforms were determined using Full Range Rainbow Molecular Weight Markers (Amersham). The fluorescence of methylumbelliferone liberated by phosphatase activity was visualized under UV light (Gel Doc 2000, ver.4.1; Bio-Rad, Hercules, CA, USA), as described before (Żebrowska, Bujnowska & Ciereszko, 2012 (link)). Protein concentration in shoot and root extracts was determined according to the Bradford (1976) (link) method, at 595 nm (CE 2501; Cecil, Cambridge, UK), with BSA as the standard.
9
Denaturing Polyacrylamide Gel Electrophoresis
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Mini gels (10 × 10 × 0.03 cm3) were prepared with shark tooth combs. The electrophoretic unit was SE260 (Hoefer, USA). The gel concentrations were 15%, 20%, 25%, 30%, 35%, 40%, and 45%. Acrylamide/bisacrylamide = 19/1. The urea concentrations in 15–35%, 40%, and 45% denaturing gels were 7, 6 and 3.5 M, respectively. Gel buffer and tank buffer were 1× and 0.5 × TBE, respectively. The sample volume was 2.5–6 μl. 0.01–10 μg of an oligo were mixed with 2 μl of formamide loading dye containing 98% (v/v) formamide, 10 mM EDTA (pH 8.0) and 0.05% (w/v) bromophenol blue, denatured at 95°C for 30 seconds, cooled on ice, and then loaded to the gels. Electrophoreses were run at room temperature. The electrophoretic voltages and times for different concentration gels were: 15%, 200 V, 1 hr; 20%, 300 V, 1 hr; 25%, 400 V, 1 hr; 30%, 500 V, 1.7–2.5 hrs; 35%, 600 V, 2–2.5 hrs; 40%, 700 V, 3.5–4 hrs; 45%, 800 V, 4.5–5 hrs.
10
Myofibrillar Protein Analysis by SDS-PAGE
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The analysis of myofibrillar protein was done by using SDS-PAGE (SE 260, Hoefer Pharmacia Biotech Inc., USA). Gel was consisted by Stacking gel 4%, separating gel 10%. The sample of myofibrillar protein was loaded 10 µg per lane, for running buffer, upper running buffer (0.1 M tris, 0.15 M glycine, 0.15% SDS) and lower running buffer (0.025 M tris, 0.2 M glycine, 0.1% SDS) were separately manufactured by modifying Laemmli (1970) (link) process and used.
The operating condition of electrophoresis running was fixed by 4℃ and 40 mA, and 2 h operated (Rivero et al., 1997 (link)). To dye gel 0.05% Coomassie blue R-250 (w/v), 40% methanol and 7% acetic acid we used for 2 h at room temperature, so that to achieve sufficient dying, and 2 times bleaching were made by 40% methanol and 7% acetic acid.
To take gel image photo of myofibrillar protein Kodak DC290 (Eastman Kodak Company, USA) was used, and to analyze gel image Kodak 1D Image Analysis Software (Eastman Kodak Company, USA) was used (Huff-Lonergan et al., 2002 ; Ryu et al., 2005 (link)).
The operating condition of electrophoresis running was fixed by 4℃ and 40 mA, and 2 h operated (Rivero et al., 1997 (link)). To dye gel 0.05% Coomassie blue R-250 (w/v), 40% methanol and 7% acetic acid we used for 2 h at room temperature, so that to achieve sufficient dying, and 2 times bleaching were made by 40% methanol and 7% acetic acid.
To take gel image photo of myofibrillar protein Kodak DC290 (Eastman Kodak Company, USA) was used, and to analyze gel image Kodak 1D Image Analysis Software (Eastman Kodak Company, USA) was used (Huff-Lonergan et al., 2002 ; Ryu et al., 2005 (link)).
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