Prior to electrophoresis, samples were incubated at 95°C for 5 min in sample loading buffer (0.25 M Tris-HCl, pH 6.8, 5% glycerol, 5% 2-mercaptoethanol, 3% SDS, and 0.2 mg/ml Bromophenol Blue), and then separated by SDS/PAGE using 10% (w/v) acrylamide in the resolving gels. The samples were resolved on SDS/PAGE gels in an electrophoresis unit (Bio-Rad), 100 V, and constant current, for 1 h, using SDS running buffer (25 mM Tris, 200 mM glycine, 0.1% SDS, pH 8.3). The total proteins were detected in the gels using Coomassie Blue staining. For Western blots, the proteins separated by electrophoresis were transferred to the PVDF membrane and incubated overnight with an anti-His-tag mouse monoclonal antibody (Genetex, 1:2000) that was used to detect FVH1-1. The blot was washed three times with TBST. An HRP-labeled secondary antibody was then added and incubated for 1 h at room temperature. The blot was washed three times with TBST, reacted with ECL Western blotting reagents for 2 min, and then were exposed by Kodak Gel Logic Imaging Station for 15–60 s.
Electrophoresis unit
Manufactured by Bio-Rad
Sourced in United States
The Electrophoresis unit is a laboratory instrument designed to separate and analyze macromolecules, such as proteins or nucleic acids, based on their size and electrical charge. It is a core tool used in various biological and biochemical applications.
Automatically generated - may contain errors
Lab products found in correlation
L2880 100mg, by Merck Group (1 mentions)
Rm2235 paraffin slicer, by Leica (1 mentions)
Dm2500 optical microscope, by Leica (1 mentions)
7100 automatic biochemical analyzer, by Hitachi (1 mentions)
Chemidoc xrs gel imaging system, by Bio-Rad (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Ecl reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Ipgphor apparatus, by GE Healthcare (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Stains all, by Merck Group (1 mentions)
Malvern zetasizer, by Malvern Panalytical (1 mentions)
Immobiline drystrip, by GE Healthcare (1 mentions)
Nanodrop one, by Thermo Fisher Scientific (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Complete mini, by Roche (1 mentions)
15 protocols using electrophoresis unit
1
Denaturing Protein Gel Electrophoresis and Western Blot
2
Endotoxin-Induced Organ Dysfunction Study
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LPS (Lipopolysaccharides from Escherichia coli O555: B5, L2880-100MG) was purchased from Sigma (USA) (Batch number: 017M4112V). Dexamethasone (DXM) was purchased from Anhui Golden Sun Biochemical Pharmaceutical Co., Ltd. (Anhui, China) (batch number: 15032521). 7100 Automatic biochemical analyzer (Hitachi, Japan), Bio-Rad electrophoresis unit (USA), and Bio-Rad ChemiDocXRS + Gel Imaging System (USA) were used in this study. LEICA RM2235 paraffin slicer (Germany) was used to generate sections for microscopy. LEICA DM2500 Optical Microscope (Germany) was used to measure neutrophil invasion. Urea nitrogen (BUN) (R1 TG836, R2 TG837), total protein (TP) (TH619), albumin (ALB) (TF126), aspartate aminotransferase (AST) (R1 AR792, R2 TG862), alanine aminotransferase (ALT) (R1 AR794, R2 TH622), lactate dehydrogenase (LDH) (R1 AR796, R2AP318) and alkaline phosphatase (ALP) (R1 TF168, R2 AR800) were all purchased from Japan Pure Pharmaceutical Industry Co., Ltd. in Shanghai, China. Creatinine (Cre) (710241 H) and creatine kinase (CK) (708021 G) were purchased from Beijing Leadman Biochemical Co., Ltd. (Beijing, China).
3
Western Blot Protein Analysis
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Following various treatments, cells were harvested by scraping and lysates were prepared as described previously [28 (link)]. Protein estimation was done by Bradford assay and 100 μg of protein samples were resolved on 12 or 15% SDS-polyacrylamide gels using a Bio-Rad electrophoresis unit. Proteins were electrophoretically transferred to PVDF membranes (Bio-Rad) for 2 h at 100 V and the membranes were blocked with 5% blocking buffer for an hour. Membranes were incubated overnight at 4°C with specific primary antibodies, as noted, washed thoroughly, then incubated with corresponding secondary antibodies conjugated with HRP. The blots were then developed with the ECL reagent (Fisher Scientific) as previously described. Protein levels were quantified by densitometry using Quantity One software (Bio-Rad).
4
Two-Dimensional Protein Separation by IEF-SDS-PAGE
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An IPGPhor apparatus (GE Healthcare, Piscataway, NJ, USA) was used for isoelectric focusing (IEF) with immobilized 13 cm (IPG) strips with a non-linear pH gradient (pH 3.0–10). The IPG strips were loaded with a 50 µg sample protein in a total volume of 250 μl of rehydration buffer [7 M urea, 2 M thiourea, 0,4 % (v/v) Triton-X100, 4 % (w/v) CHAPS, 1 % (v/v) IPG buffer, 60 mM DTT]. The voltage settings for the IEF were 30 V, 12 h (re-hydration step); 100 V, 150 Vh (S2); 250 V, 250 Vh (S3); 1,000 V, 1,500 Vh (S4); 2,500 V, 2,500 Vh (S5); 8,000 V, 30 min (Gradient, S6); 8,000 V, 32,000 Vh (S7); 100 V (S8).
Following IEF, the gel strips were denatured twice (50 mM Tris–HCl pH 8.8, 6 M urea, 30 % glycerol, 2 % SDS) over a period of 15 min, first with 1 % DTT followed by a second incubation with the same buffer containing 2.5 % iodoacetamide.
The second dimension electrophoresis was performed on a 12.5 % gel using an electrophoresis unit (Bio-Rad Laboratories Inc.). Gels were stained with Flamingo reagent (Bio-Rad Laboratories Inc.). Briefly, each gel was fixed with 200 ml of 40 % Ethanol, 10 % acetic acid solution and stained with 100 ml of 1/10 diluted Flamingo reagent. Stained gels were scanned on a Fujifilm FLA-5100 (Fuji Storm) using a 532 nm laser at 600 V.
Following IEF, the gel strips were denatured twice (50 mM Tris–HCl pH 8.8, 6 M urea, 30 % glycerol, 2 % SDS) over a period of 15 min, first with 1 % DTT followed by a second incubation with the same buffer containing 2.5 % iodoacetamide.
The second dimension electrophoresis was performed on a 12.5 % gel using an electrophoresis unit (Bio-Rad Laboratories Inc.). Gels were stained with Flamingo reagent (Bio-Rad Laboratories Inc.). Briefly, each gel was fixed with 200 ml of 40 % Ethanol, 10 % acetic acid solution and stained with 100 ml of 1/10 diluted Flamingo reagent. Stained gels were scanned on a Fujifilm FLA-5100 (Fuji Storm) using a 532 nm laser at 600 V.
5
Agarose Gel Electrophoresis of mRNA-hLNCs
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Agarose
gel electrophoresis was performed by using 1.0% (w/v) agarose gel.
Briefly, agarose gel was prepared in Tris base, acetic acid, and EDTA
(TAE) buffer containing 0.5 μg/mL of EtBr. Free mRNA and/or
mRNA-hLNCs complex was loaded into wells along with loading dye (Bromophenol
blue, 6X). It was electrophoresed using an electrophoresis unit (Bio-Rad,
USA) at 80 V for 15–20 min. The mRNA was tracked using a ChemiDoc
Imaging System (Bio-Rad)
gel electrophoresis was performed by using 1.0% (w/v) agarose gel.
Briefly, agarose gel was prepared in Tris base, acetic acid, and EDTA
(TAE) buffer containing 0.5 μg/mL of EtBr. Free mRNA and/or
mRNA-hLNCs complex was loaded into wells along with loading dye (Bromophenol
blue, 6X). It was electrophoresed using an electrophoresis unit (Bio-Rad,
USA) at 80 V for 15–20 min. The mRNA was tracked using a ChemiDoc
Imaging System (Bio-Rad)
6
Characterization of siRab26-DYM Nanovector
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The siRab26-DYM nanovector was analyzed by native polyacrylamide gel electrophoresis (PAGE). The samples were run on gels containing 6% polyacrylamide (19:1 acrylamide: bisacrylamide) in a Bio-Rad electrophoresis unit at 4°C (120 V, constant voltage) with 1× TAE/Mg2+ running buffer. After electrophoresis, the gels were stained with 0.01% 'Stains-All' (Sigma) for 1 h and scanned.
Moreover, the siRab26-DYM nanovector was analyzed using dynamic light scattering (DLS) measurements. The siRab26-DYM solution was diluted to ~50 nM and measured with a Malvern Zetasizer (Malvern Instruments Ltd, UK) in 1× TAE/Mg2+ buffer. Pure 1× TAE/Mg2+ was used as the blank.
Moreover, the siRab26-DYM nanovector was analyzed using dynamic light scattering (DLS) measurements. The siRab26-DYM solution was diluted to ~50 nM and measured with a Malvern Zetasizer (Malvern Instruments Ltd, UK) in 1× TAE/Mg2+ buffer. Pure 1× TAE/Mg2+ was used as the blank.
7
Agarose Gel Electrophoresis Protocol
8
Two-Dimensional Electrophoresis of Flagellar Proteins
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Two-dimensional (2D) electrophoresis was performed as described by Görg et al [46] (link). A total of 120 µg of desalted flagellar proteins solubilized in 8 M urea containing 2% (v/v) CHAPS and 20 mM DTT was loaded onto an immobilized pH gradient IPG gel strip 3–11 NL (Immobiline Dry strip, GE HealthCare) and isoelectrofocused subsequently at 500 V for 30 min, 1000 V for 1 h, 2000 V for 1 h, and 5000 V for 7 h. The strips were incubated in equilibration buffer (75 mM Tris–HCl pH 6.8, 6 M urea, 30% (v/v) glycerol, 2% (w/v) SDS, 0.01% (w/v) bromophenol blue) containing 10 mg/ml DTT for 15 min and then in equilibration buffer containing 2.5 mg/ml iodoacetamide for 15 min. Equilibrated strips were placed on 12% polyacrylamide gels, sealed with 0.5% agarose solution, and proteins separated at 4 °C using a Bio-Rad electrophoresis unit. Gels were either blotted onto nitrocellulose membranes for immunoblot analysis as indicated above or stained with colloidal Coomassie Blue G-250. Individual spots were excised from the gel and submitted to the Centro de Estudios Químicos y Biológicos, Universidad de Buenos Aires, Buenos Aires, Argentina for protein identification by MALDI-TOF mass spectrometry. MS/MS spectra were analysed as described previously.
9
SDS-PAGE Protein Purity Analysis
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SDS-PAGE electrophoresis (Bio-Rad electrophoresis unit) was performed according to the method of Laemmli [28] by using 12 % SDS-polyacrylamide gels to determine the purity and approximate molecular mass of the enzyme. All proteins run on the polyacrylamide gel were stained with 0.2% (w/v) Coomassie brilliant blue R-250 for 1 h, then washed with the SDS-PAGE destaining solution (100 mL acetic acid, 300 mL methanol, 600 mL dH 2 O) to remove excess dye.
10
SDS-PAGE Analysis of Insulin-Albumin Bioconjugate
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Size of the modified bioconjugate
was determined
using SDS-PAGE run in the Bio-Rad Electrophoresis Unit. The concentrated
insulin–albumin conjugate was subjected to SDS-PAGE (8%) and
a constant current of 120 V was applied to the gel. The size of the
bioconjugate was compared with a known standard marker.
was determined
using SDS-PAGE run in the Bio-Rad Electrophoresis Unit. The concentrated
insulin–albumin conjugate was subjected to SDS-PAGE (8%) and
a constant current of 120 V was applied to the gel. The size of the
bioconjugate was compared with a known standard marker.
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