The electrophoretogram of WJSP and WJSPHs was measured according to the previous report (Jiang et al., 2018 (link)). Briefly, SDS– PAGE electrophoresis used 12.5% separating gel and 5% stacking gel, and Tricine– SDS– PAGE electrophoresis used 16.5% separating gel, 10% sandwich gel, and 4% stacking gel. The sample solution was blended with loading buffer in same volume, and 10 µl of the mixed solution was added into the gel. SDS– PAGE electrophoresis was carried out by Bio‐Rad electrophoresis apparatus, and the samples were run at 70 V for 30 min and subsequently at 140 V for 1 h. Tricine– SDS– PAGE electrophoresis was carried out by Bio‐Rad electrophoresis apparatus, and all samples were allowed to run at 30 V for 1 h and subsequently at 100 V for 5 h. Finally, the obtained gels were dyed in Coomassie Bright Blue solution and destained in a mixed solution (glacial acetic acid:methanol:water = 1:3:6).
Electrophoresis apparatus
Manufactured by Bio-Rad
Sourced in United States
An electrophoresis apparatus is a laboratory equipment used to separate and analyze macromolecules, such as DNA, RNA, or proteins, based on their size, charge, or other physical properties. It provides a controlled environment for the electrophoretic separation process.
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Lab products found in correlation
Pvdf membrane, by Merck Group (7 mentions)
Chemidoc, by Bio-Rad (6 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Quickblock, by Beyotime (4 mentions)
Skim milk, by Merck Group (4 mentions)
Supersignal west pico plus chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Ibfp0785c, by Bio-Rad (3 mentions)
Semi dry transfer instrument, by Bio-Rad (3 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (3 mentions)
Ecl imaging system, by Bio-Rad (3 mentions)
Chemidoc xrs plus gel documentation system, by Bio-Rad (2 mentions)
O generuler dna ladder mix, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Bio-Rad (2 mentions)
Gel imaging system, by Bio-Rad (2 mentions)
Ab32503, by Abcam (2 mentions)
Ripa lysis solution, by Beyotime (2 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Gel image analysis system, by Bio-Rad (2 mentions)
Erk1 2, by Merck Group (2 mentions)
69 protocols using electrophoresis apparatus
1
Electrophoresis of Protein Samples
2
SDS-PAGE Fractionation of Mosquito SGE
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1-DE experiments were performed for fractionation of SGE samples of SF mosquitoes. Briefly, 27 μg of SGE sample was mixed with sample buffer (0.625M TrisHCl, 10% SDS, glycerol, and distilled water) containing β-mercaptoethanol (10% vol/vol) and boiled for 5 min at 95°C. Denatured protein sample were then loaded on to SDS PAGE mini gel consisting of 3% stacking and 12% resolving gel of 1-mm thickness along with the protein molecular weight marker (Genei protein range marker, Bangalore Genei) and subjected to electrophoresis (Bio-Rad electrophoresis apparatus, USA). After completion of the run, the gel was stained with FOCUS-FAST silverTM stain (G-Biosciences). Various bands were observed on the gel after silver staining. Each band was cut and collected in separate eppendorf tube in 50 μl of stop solution (2% acetic acid) and further stored at -20°C for trypsin digestion.
3
Optimizing Emulsion PCR Conditions
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The reagents in the experiment included Span 80, Tween 80, and mineral oil (Sigma, St. Louis, MO, USA), and Pfu DNA polymerase and dNTP (Sangon, Shanghai, People's Republic of China). An MJ‐Mini PCR amplification system (Bio‐Rad, CA, USA), a Bio‐Rad electrophoresis apparatus (Bio‐Rad), and a magnetic stirrer (VWR, West Chester, PA, USA) were used in the experiment.
4
Protein Expression in Sertoli-Germ Cells
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Total protein was extracted from the testes of rats and co-cultured Sertoli–germ cells exposed to different concentrations of MC-LR with or without RES. Samples containing 30 µg of protein underwent electrophoresis with a Bio-Rad electrophoresis apparatus (Bio-Rad, Hercules, CA, USA), were separated by 12% SDS-PAGE, and subsequently transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The membranes were blocked in tris buffered saline with tween (TBST) containing 5% BSA at 23 °C for two hours, and immunoblotted using primary anti-Ku70 (sc-17789, Santa Cruz Biotechnology, Boston, CA, USA), anti-SIRT1 (ab110304), anti-p53 (ab131442), anti-p53 (acetyl K381, ab61241), anti-cleaved-caspase-3 (ab2302), anti-Bax (ab32503), anti-Bcl-2 (ab7973), and anti-β-actin (ab6276) (Abcam, Cambridge, UK). An enhanced chemiluminescence detection kit (Beijing ComWin Biotech Co., Ltd., Beijing, China) was used to analyze the protein bands. The intensity of the bands was quantified using the Bio-Rad Quantity One software.
5
Western Blotting Protein Extraction
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Total protein from tissues and cells was extracted using RIPA lysis buffer (P0013B; Beyotime Biotechnology, China) containing PMSF. The protein concentration was determined using the Bradford method. Equal amounts of protein samples were resolved and separated by 10% SDS-PAGE using an electrophoresis apparatus (Bio-Rad, America) and transferred onto polyvinylidene di uoride (PVDF) membranes. Next, the membranes were blocked by incubating with Quick Block (P0252; Beyotime Biotechnology, China) for 20 minutes. Next, the membranes were treated with primary antibody, using GAPDH as an internal reference, at 4°C overnight. Finally, the membranes were washed and then incubated with secondary antibody for 2 h at room temperature. The blots were then visualized by enhanced chemiluminescence detection.
6
SDS-PAGE Analysis of Protein Molecular Weights
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The molecular weight (Mw) distribution of Mb, Hb and MP was determined by SDS-PAGE. Mw was determined by using proteins markers (TransGen Biotech, Beijing, China) with molecular weights of 14–120 kDa and 15–250 KDa, according to the Mw of Mb (17.6 KDa) and Hb (64.5 KDa). Electrophoresis was performed at 100 V with a Bio-Rad electrophoresis apparatus (Richmond, CA, USA) with 5% concentrated gel and 12.5% separated gel. The bands were shown by Coomassie Brilliant Blue R-250 (Xia, et al., 2009 (link)).
7
Gel Electrophoresis of Annealed Samples
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In all, 10 μL of each annealed sample was subjected to the 8% PAGE (19:1 acrylamide/bisacrylamide in 1 × TAE/Mg2+ buffer). Gels were run at 100 V (constant voltage) for about 2 h with electrophoresis apparatus (Bio-Rad, United States). Then, the gels were stained with 1 × GelRed nucleic acid dye (Biotium, United States) and scanned under UV light.
8
Western Blotting Protein Extraction
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Total protein from tissues and cells was extracted using RIPA lysis buffer (P0013B; Beyotime Biotechnology, China) containing PMSF. The protein concentration was determined using the Bradford method. Equal amounts of protein samples were resolved and separated by 10% SDS-PAGE using an electrophoresis apparatus (Bio-Rad, America) and transferred onto polyvinylidene di uoride (PVDF) membranes. Next, the membranes were blocked by incubating with Quick Block (P0252; Beyotime Biotechnology, China) for 20 minutes. Next, the membranes were treated with primary antibody, using GAPDH as an internal reference, at 4°C overnight. Finally, the membranes were washed and then incubated with secondary antibody for 2 h at room temperature. The blots were then visualized by enhanced chemiluminescence detection.
9
Nanoplexes for Oligonucleotide Delivery and Stability
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Nanoplexes were synthesized through direct mixing of oligonucleotide sequences (As-21 or Scr) with PEG5k-CMPEI NG at different N/P ratios. The product was further incubated for 0.5, 3, 6, and 24 hours at 25°C. Agarose gel retardation assay was conducted using a Bio-Rad electrophoresis apparatus at 60 V, for 30 minutes in 2.5% (w/v) agarose gel, and visualized on a UV trans-illuminator. For investigating the biological stability against nucleases, As-21/NG Nanoplexes (containing 3 μg DNA at the N/P=5) were incubated with 2 μL 10X reaction buffer and 2 U of DNase I. The mixture was then incubated at 37ºC for 1, 3, 6, and 24 hours. An aliquot was run at 2.5% agarose gel electrophoresis and its stability against DNase I was compared with the naked As-21. EthBr dye exclusion assay was carried out to assess the stability of As-21/NG nanoplexes against polyanionic heparin sulfate simulating the extracellular matrix. Briefly, to each well of a 96-well plate containing nanoplexes with various N/P ratios, 1 IU heparin sulfate was added per µg of the oligonucleotide. Following 15-minute incubation in dark, 50 μL EthBr (1 µg/mL) was added and fluorescence intensity was measured at the respective excitation and emission wavelengths of λ = 510 and 595 nm. Oligonucleotide efflux (%) was calculated from fluorescence intensities prior to and after adding heparin sulfate.
10
Optimizing Nanoparticle Binding Ratios
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The optimum binding ratios (w/w) for the FAuNPs were assessed using a gel retardation assay [27 (link)]. Nanocomplexes were prepared as in Section 4.5 . and Table 3 . Gel loading buffer (40% sucrose, 0.5% bromophenol Blue, 2 µL) was added to the nanocomplexes, which were then subjected to agarose gel electrophoresis for 60 min at 50 volts using a 1% agarose gel in a Bio-Rad electrophoresis apparatus containing 1× electrophoresis buffer (36 mM Tris-HCl, 30 mM sodium phosphate, 10 mM EDTA pH 7.5) and 3 µL of ethidium bromide (10 mg/mL). The resultant gel was viewed under UV transillumination at a 300 millisecond exposure time in a Vacutec Syngene G:Box BioImaging system (Syngene, Cambridge, UK).
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