Cells were lysed in the SDS/Nonidet P-40 lysis buffer [1% SDS, 1% Nonidet P-40, 50 mM Tris (pH 8.0), 150 mM NaCl, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 mM NaF, and 100 µM Na3VO4]. Nucleus and cytosol protein fractions were extracted using Nuclear/Cytosol Fractionation kit (BioVision). The lysates were boiled for 5 min and then cleared by centrifugation at 15,000 rpm and 4°C. A Bradford protein assay reagent (BioRad) was used to determine the protein concentration of supernatants. Lysates were further boiled for 5 min in the sample buffer. Then, samples were resolved using the SDS-PAGE and transferred onto Immobilon-P (Millipore Corp.) sheets. Blots were first incubated in blocking buffer [5% (w/v) nonfat dry milk in Tris-buffered saline (TBS) plus 0.05% Tween 20] for 30 min. Then, they were incubated with a primary antibody for 16 h at 4°C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 30 min at room temperature (RT). The ECL-plus chemiluminescence (GE Healthcare) was used to visualize antibody-antigen complex.
Ecl plus chemiluminescence
Manufactured by GE Healthcare
Sourced in United Kingdom
ECL-plus chemiluminescence is a laboratory equipment used for the detection and quantification of proteins in Western blot analysis. It generates a luminescent signal that can be captured and analyzed to determine the presence and abundance of specific proteins within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p, by Merck Group (3 mentions)
Bradford protein assay reagent, by Bio-Rad (2 mentions)
Nuclear cytosol fractionation kit, by Abcam (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Protein assay dye reagent concentrate, by Bio-Rad (1 mentions)
Anti bmal1, by Abcam (1 mentions)
Complete mini edta free, by Roche (1 mentions)
Anti clock, by Abcam (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Agilent Technologies (1 mentions)
Phosphor p38 mapk, by Cell Signaling Technology (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Pierce bca method, by Thermo Fisher Scientific (1 mentions)
Phosphor p44 42, by Cell Signaling Technology (1 mentions)
5 protocols using ecl plus chemiluminescence
1
Subcellular Protein Fractionation and Western Blotting
2
Western Blot Analysis of Circadian Clock Proteins
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Whole-cell protein extracts were prepared by lysing cells in RIPA buffer containing protease inhibitors (cOmplete, Mini, EDTA-free; Roche, Welwyn Garden City, United Kingdom). Protein yield was quantified using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hemel Hempstead, United Kingdom). Equal amounts of protein were separated by SDS-PAGE before wet transfer onto PVDF membrane (GE Healthcare, Buckinghamshire, United Kingdom). Nonspecific binding sites were blocked by overnight incubation with 5% nonfat dry milk in Tris-buffered saline with 1% Tween 20 [130 mM NaCl, 20 mM Tris (pH 7.6), and 1% Tween 20]. The following primary antibodies were purchased from Abcam (Cambridge, United Kingdom): anti-CLOCK (catalog number ab3517, diluted 1:3000); anti-BMAL1 (ab3350, 1:375); anti-CRY1 (ab54649, 1:500); anti-CRY2 (ab38872, 1:2000); anti-PER1 (ab3443, 1:300); anti-PER2 (ab179813, 1:300); and anti-β-actin (ab8226, 1:10,000). Protein complexes were visualized with ECL Plus chemiluminescence (GE Healthcare). The Western blots are collated in Supplemental Fig. 1 .
3
Protein Extraction and Western Blotting
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Cells were lysed in SDS/Nonidet P-40 lysis buffer (1% SDS, 1% Nonidet P-40, 50 mM Tris [pH 8.0], 150 mM NaCl, 2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride [PMSF], 5 mM NaF, 100 μM Na3VO4). The lysates were boiled for 5 min and then cleared by centrifugation at 15,000 rpm and 4 °C. Protein concentration of the supernatant was determined using a Bradford protein assay reagent (BioRad). The lysates were further boiled for 5 min in sample buffer. Samples were then resolved by SDS-PAGE and transferred onto Immobilon-P (Millipore Corp.) sheets. The blots were first incubated in blocking buffer (5% [w/v] nonfat dry milk in Tris-buffered saline [TBS] plus 0.05% Tween 20) for 1 h. The blots were then incubated with a primary antibody for 16 h at 4 °C, followed by incubation with a horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The antibody-antigen complex was visualized by ECL-plus chemiluminescence (GE Healthcare).
4
Quantifying Yeast RNA Polymerase II
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Yeast cultures were grown in synthetic defined medium with or without DOX (50 μg/ml) from a starting A600 of between 0.1 and 0.2 and grown to mid-log phase (A600 0.6–1.0). In all cases, five optical units of cells were harvested by centrifugation, and extracts were prepared as previously described (19 (link)). For Western analysis of Rpb1p, proteins were separated on 8% SDS-PAGE gel made with 19:1 acrylamide:bis-acrylamide. Gels were transferred to PVDF for 90 min at 45 mA and dried in methanol to block according to the manufacturer's instructions (Millipore-Immobilon-P). Dried membranes were rehydrated briefly in methanol and incubated with primary antibodies (Y-80; Santa Cruz) overnight at 4 °C in PBST (phosphate-buffered saline including 0.05% Tween 20) containing 4% milk. Western blots were visualized using HRP-conjugated secondary antibodies and ECL Plus chemiluminescence (GE Healthcare).
5
AGEs-induced MAPK Signaling Regulation
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Growth-arrested PTECs were stimulated with AGEs (100 µg/mL) with or without BMP7 (100 ng/mL) for 30 min. Cells were washed with PBS and then lysed with lysis buffer containing protease inhibitor (Sigma Aldrich) as previously described [33] . Total protein concentrations were determined by Pierce BCA method (Thermo Scientific, Rockford, USA). Equal amount of proteins were dissolved in loading buffer and denatured by boiling at 95°C for 10 min. Protein samples were run on 12% SDS-PAGE gel and transferred to PVDF membrane (Millipore).
Membrane were blocked with 5% non-fat milk and incubated overnight with primary antibodies against phosphor-p38 MAPK (1:1000), p38 MAPK (1:2000) , phosphor-p44/42 (1:2000) and p44/42 (1:2000) (Cell Signaling Technology, Beverly, MA) in Tris-buffered saline with 0.1% Tween-20, and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (Dako, Carpinteria, CA) for 2 h. Reactions were visualized by using ECL plus chemiluminescence (GE Healthcare, Buckinghamshire, UK) with ChemiDoc XRS+ system (Bio-Rad, Hercules, CA). Density of bands were quantified by bundled ChemiDoc software and normalized by actin level.
Membrane were blocked with 5% non-fat milk and incubated overnight with primary antibodies against phosphor-p38 MAPK (1:1000), p38 MAPK (1:2000) , phosphor-p44/42 (1:2000) and p44/42 (1:2000) (Cell Signaling Technology, Beverly, MA) in Tris-buffered saline with 0.1% Tween-20, and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies (Dako, Carpinteria, CA) for 2 h. Reactions were visualized by using ECL plus chemiluminescence (GE Healthcare, Buckinghamshire, UK) with ChemiDoc XRS+ system (Bio-Rad, Hercules, CA). Density of bands were quantified by bundled ChemiDoc software and normalized by actin level.
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