Native polyacrylamide gel electrophoresis (PAGE) was performed using ReadyGels (7.5%; Bio-Rad) as described in our previous study (23 (link)). Briefly, gels were prerun with 25 mM Tris and 192 mM glycine (pH 8.4) with 1% deoxycholate (DOC) in the cathode chamber for 30 min at 40 mA. Samples in native sample buffer (10 μg protein, 62.5 mM Tris-Cl [pH 6.8], 15% glycerol, and 1% DOC) were size fractionated by electrophoresis at 80 V and transferred to PVDF membranes for WB analysis.
Ready gel
Manufactured by Bio-Rad
Sourced in United States
Ready Gel is a pre-cast polyacrylamide gel for electrophoresis. It is available in a variety of formats, including different gel percentages and well configurations, to accommodate various sample separation needs.
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Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Supersignal west femto kit, by Thermo Fisher Scientific (2 mentions)
Img 5142a, by Novus Biologicals (2 mentions)
Las 4000, by Fujifilm (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Memcode reversible stain, by Thermo Fisher Scientific (2 mentions)
Immobilon transfer membrane, by Merck Group (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Pageruler plus prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Mini protean tetra cell, by Bio-Rad (1 mentions)
Anti notch1, by Abcam (1 mentions)
Anti notch3, by Abcam (1 mentions)
Anti notch2, by Abcam (1 mentions)
Anti dll4, by Abcam (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Ab9484, by Abcam (1 mentions)
Ab95950, by Abcam (1 mentions)
Af3925, by R&D Systems (1 mentions)
Quant it protein assay kit, by Thermo Fisher Scientific (1 mentions)
Amersham ecl plus western blotting detection system, by GE Healthcare (1 mentions)
30 protocols using ready gel
1
Native PAGE for Protein Separation
2
Detecting Genetic Differences via Heteroduplex Analysis
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For HMA, 4.5 µl aliquots of PCR amplified products of two clones (a reference clone and a test clone within each sub-isolate) were combined, and 1 µl of 10X annealing buffer (100 mM Tris-HCl, pH 8.0 containing 1M NaCl and 20 mM EDTA) was added. The DNA mixture was denatured at 95°C for 10 min, reannealed at 68°C for 1 hr, incubated at 0°C for 10 min, and then electrophoresed on a 10% polyacrylamide gel (Bio-Rad ready gels) for 2.5 hrs at 120 volts at 4°C. Heteroduplexes, when present, migrate between the lower band of homoduplex DNA and the upper band of ssDNA. The heteroduplexes indicate that the test clone is genetically different from the reference clone (Figure 1
3
SDS-PAGE Analysis of IFNβ-1a Proteins
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Electrophoresis was performed on a polyacrylamide gel cross-linked with 40: 1 methylenbisacrylamide with sodium dodecyl sulphate and measured by electrophoresis system Mini-PROTEAN® Tetra cell (Bio-Rad, Gladesville, Australia) according to a previously described protocol [38 (link)]. The IFNβ-1a solution was heated at 100 °C for 5 min and electrophoresis under non-reducing conditions (20 ng IFNβ-1a protein-loaded or PageRuler™ plus prestained protein ladder (Thermo Fisher Scientific)) using 4% Tris hydrochloride [39 (link)]. Gradient polyacrylamide gels (Bio-Rad Ready Gels) for 1 h. Eighty μL of IFNβ-1 and 20 μL of 5× Sample Buffer solution, in each well contained 2.63 μg of IFNβ-1a Rebif or ReciGen that corresponded to 25 μL of solution. The electrophoresis was carried out applying 40 mA with voltage of 150 V. The staining of proteins was carried out using Coomassie blue R250. A more precise technique is described in the source [39 (link)].
4
Western Blot Analysis of Notch Signaling
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The protein samples were analyzed using a BCA kit (Pierce Company). The samples were separated by 10–20% Ready Gels (Bio-Rad, USA) and transferred onto a polyvinylidene fluoride (PVDF) membrane for 30 min in a transfer electricity meter (Bio-Rad, USA). The PVDF nitrocellulose films were cleared and sealed with 5% milk. After washing, anti-Notch 1 (1: 500, Abcam, USA), anti-Notch 2 (1: 800, Abcam, USA), anti-Notch 3 (1: 1000, Abcam, USA), and anti-DLL4 (1: 1000, Abcam, USA) were added to incubate overnight at 4°C. Then, these films were washed 3 times with Tris-buffered saline tween (TBST) and incubated with HRP secondary antibodies (1: 5000, Sigma, USA) at room temperature for 1 h. Relative expression levels of each protein were normalized to endogenous control β-actin using Image J software.
5
SDS-PAGE Protein Separation and Quantification
6
Immunoblotting of Mesenchymal Stem Cells
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Immunoblotting was performed as previously described.11 (link),20 (link) Briefly, MSC were plated
at 5 × 104 cells per well in a six-well plate prior to lysis with
universal lysis buffer (Millipore). Protein quantification was performed with
Qubit Fluorometer and Quant-iT™ protein assay kit (Invitrogen) to ensure equal
loading of samples. Protein lysates were diluted 1:1 with 2 × Laemmli buffer and
denatured at 95°C, before loading on Tris HCl 4–20% ready gels (Bio-Rad). Gels
were transferred to nitrocellulose membrane and subsequently blocked with 5%
bovine serum albumin (Sigma) or 5% milk in tris-buffered saline–Tween for 1
hour. Incubation with primary antibody anti-FH (Abcam, ab95950),
anti-nuclear-related (erythroid-derived 2) factor 2 (anti-NRF-2) antibody
(R&D, AF3925), anti-hypoxia inducible factor1α (anti-hypoxia inducible
factor1α (anti-HIF-1α; Abcam, ab51608)), anti-COX5 (Santa Cruz, SC-376907) and
anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam, ab9484) was
performed overnight at 4°C. Amersham ECL Plus™ Western Blotting Detection System
(GE Healthcare) was used to visualise specific protein expression patterns by
chemiluminescence. The integrated density of bands was measured using ImageJ
(National Institute of Health, NIH), and values are expressed relative to GAPDH
loading control protein.
at 5 × 104 cells per well in a six-well plate prior to lysis with
universal lysis buffer (Millipore). Protein quantification was performed with
Qubit Fluorometer and Quant-iT™ protein assay kit (Invitrogen) to ensure equal
loading of samples. Protein lysates were diluted 1:1 with 2 × Laemmli buffer and
denatured at 95°C, before loading on Tris HCl 4–20% ready gels (Bio-Rad). Gels
were transferred to nitrocellulose membrane and subsequently blocked with 5%
bovine serum albumin (Sigma) or 5% milk in tris-buffered saline–Tween for 1
hour. Incubation with primary antibody anti-FH (Abcam, ab95950),
anti-nuclear-related (erythroid-derived 2) factor 2 (anti-NRF-2) antibody
(R&D, AF3925), anti-hypoxia inducible factor1α (anti-hypoxia inducible
factor1α (anti-HIF-1α; Abcam, ab51608)), anti-COX5 (Santa Cruz, SC-376907) and
anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Abcam, ab9484) was
performed overnight at 4°C. Amersham ECL Plus™ Western Blotting Detection System
(GE Healthcare) was used to visualise specific protein expression patterns by
chemiluminescence. The integrated density of bands was measured using ImageJ
(National Institute of Health, NIH), and values are expressed relative to GAPDH
loading control protein.
7
Native Polyacrylamide Gel Electrophoresis
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Native polyacrylamide gel electrophoresis (PAGE) was performed using ReadyGels (7.5%, Bio-Rad). In brief, gels were prerun with 25 mM Tris and 192 mM glycine, pH 8.4, with 1% deoxycholate (DOC) in the cathode chamber for 30 min at 40 mA. Samples in native sample buffer (10 g of protein, 62.5 mM Tris–Cl, pH 6.8, 15% glycerol, and 1% DOC) were size-fractionated by electrophoresis at 25 mA, and the gel was transferred to nitrocellulose membranes for WB analysis.
8
Western Blot Analysis of Homer1a
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Western blot was done as described (Sun et al., 2011 (link)). Briefly, cingulate cortex tissue was homogenized in cold buffer (lithium lauryl sulfate, 2%; aprotinin, 1.7 mg/ml; phenylmethylsulfonyl fluoride, 10 mg/ml; sodium orthovanadate, 1 mM). The homogenate was centrifuged at 15,000 rpm for 15 min at 4 °C. Protein concentration was determined by the Bradford method. Protein samples (40 μg) were analyzed by SDS-PAGE with 5–15% or 10–20% Ready Gels (Bio-Rad). After blocking with a skim milk solution (5%), immunoblotting was done with goat anti-Homer1a (1:500; SC8922; Santa Cruz Biotechnology) and rabbit anti-β-actin antibodies (1:2000; IMG-5142A; Imgenex). After reaction with HRP-conjugated secondary antibodies (donkey anti-goat IgG, 1:1000. HAF109, R&D Systems; goat anti-rabbit IgG, 1:1000, catalog no. 32460, Thermo Scientific), bands were detected with a chemiluminescence substrate kit (Super-Signal West Femto kit, Thermo Scientific) and a detector (LAS-4000, FUJIFILM) and analyzed by using ImageJ software.
9
Glutaraldehyde Cross-linking Protein Assay
10
Western Blot Protein Analysis Procedure
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Cells were washed with ice-cold phosphate-buffered saline and trypsinized and lysis buffer (Intron, Seoul, Korea) was added. The lysates were stored at −20 °C until analysis. The amount of protein was quantified by the Bradford assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein were loaded onto Readygels (4%–20% Tris-HCL; Bio-Rad) and electrophoresis was performed according to the manufacturer's instructions. Proteins were blotted onto polyvinylidene fluoride (PVDF) membranes (Invitrogen, Carlsbad, CA, USA) and incubated for 1 h at room temperature in 5% skim milk for blocking. Blots were incubated with primary antibodies overnight at 4 °C and with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The membranes were developed using enhanced chemiluminescence.
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