Frozen hearts were homogenized in RIPA buffer (Thermo Fisher Scientific, Massachusetts, USA) containing plus proteinase inhibitors and phosphatase inhibitors (Roche, Basel, Switzerland) and centrifuged at 20,000 x g for 10 min at 4°C. After a determination of protein concentrations, SDS-PAGE was performed using the supernatant with CriterionTM Precast Gels (10–20% Tris-Tricine/Peptide, BIO-RAD, California, USA) for PLN and CriterionTM TGXTM Precast Gels (4–20%, BIO-RAD) for SERCA2a and actin. They were transferred to PVDF membranes in the Trans-Blot Turbo (BIO-RAD) and blocked in PVDF Blocking Reagent (TOYOBO, Osaka, Japan). For immunoreaction, blots were incubated with 1: 1000 diluted monoclonal antibody to PLN (MilliporeSigma, Darmstadt, Germany), 1: 1000 diluted monoclonal antibody to SERCA2a (abcam, Cambridge, UK), or 1: 1000 diluted monoclonal antibody to actin (MilliporeSigma) at 4°C overnight. The antigens were detected by the luminescence method (Amersham ECL Prime kit, GE Healthcare, Little Chalfont, UK) with 1: 25000 diluted anti-mouse IgG (GE Healthcare) or 1: 25000 diluted anti-rabbit IgG (GE Healthcare) in the ChemiDoc (BIO-RAD). The intensity of bands was calculated by Image Lab 4.0 (BIO-RAD).
Criterion tgxtm precast gel
Manufactured by Bio-Rad
Sourced in United States
The Criterion™ TGX™ Precast Gels are a line of pre-made polyacrylamide gels designed for electrophoretic separation of proteins. These gels provide a consistent and reliable platform for protein analysis and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pvdf blocking reagent, by Toyobo (1 mentions)
Amersham ecl prime kit, by GE Healthcare (1 mentions)
Image lab 4, by Bio-Rad (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
Sypro ruby, by Bio-Rad (1 mentions)
Pro q diamond, by Bio-Rad (1 mentions)
Pro q diamond, by Thermo Fisher Scientific (1 mentions)
Sypro ruby, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Multi gauge v3, by Fujifilm (1 mentions)
Lactate dehydrogenase, by Merck Group (1 mentions)
Nω nitro l arginine methyl ester hydrochloride l name, by Merck Group (1 mentions)
Irdye 680rd goat anti rabbit igg, by LI COR (1 mentions)
S nitrosoglutathione, by Cayman Chemical (1 mentions)
Dulbecco s modification of eagle s medium dmem, by Corning (1 mentions)
Irdye 800cw goat anti mouse igg, by LI COR (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
6 protocols using criterion tgxtm precast gel
1
Quantification of PLN, SERCA2a in Heart
2
Phosphorylation Analysis of Myofilament Proteins
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To analyze the phosphorylation status of the myofilament proteins cMyBPC and cTnI, we used the dual staining system ProQ-Diamond/SYPRO-Ruby (Thermo Fisher Scientific) according to manufacturers’ instructions. Homogenized LV samples were separated on 4–15% CriterionTMTGXTM precast gels (BioRad, Munich, Germany) and stained with ProQ-Diamond for 1 h and subsequently with SYPRO Ruby overnight. Proteins were visualized using the ChemiDoc imaging system (BioRad). Stained protein bands were quantified via densitometry using the Multi Gauge V3.2 software (FUJIFILM Corp, Minato, Tokyo, Japan). Phospho-signals on ProQ-Diamond-stained gels were normalized to the corresponding myosin heavy chain (MHC) total protein signal on SYPRO-Ruby stained gels.
3
Cellular Signaling Pathway Protocol
4
Western Blot Analysis of Frozen Brain Samples
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Frozen brain samples were homogenised in homogenising buffer containing NP-40 cell lysis buffer (Invitrogen, #FNN0021), Protease Inhibitor Cocktail (Sigma-Aldrich, #P8340), β-Glycerophosphate (Sigma-Aldrich, #G9422), and PMSF (Sigma-Aldrich, #P7626). Protein concentration of each sample was measured by the DC Protein Assay (Bio-Rad, #500-0111). Western blot experiments were performed as described previously (Pan et al., 2016b; Pan et al., 2016a; Pan et al., 2016c) . Briefly, each sample containing 10µg of protein was denatured, and loaded into Criterion TM TGX TM Precast Gels (Biorad, #5671095). The proteins were separated in Criterion TM Vertical Electrophoresis Cells (Bio-rad, #1656001), and then electrophoretically transferred to a polyvinylidene difluoride membrane in Criterion TM Blotters (Bio-rad, #1704071). All membranes were then blocked by 5% skim milk powder, and incubated in primary antibodies and secondary antibodies, respectively. The immunoreactive bands were visualised using Amersham Hyperfilm ECL (GE Healthcare, #28-9068-36) and Luminata Classico Western HRP substrate (Millipore, #WBLUC0500). All Western blot experiments were performed in duplicate to ensure consistency.
The following antibodies were used to detected corresponding proteins: anti-PKA-Cα
The following antibodies were used to detected corresponding proteins: anti-PKA-Cα
5
Kidney Protein Expression Analysis
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The right kidney was harvested immediately after euthanasia, and a quarter of the kidney was minced and digested using the RIPA lysis buffer supplemented with 100 μg PMSF and 1 tablet of protease/phosphatase inhibitor (Thermo Fisher Scientific) per 10 ml buffer. After measuring concentrations using a modified DCTM protein assay, equal amounts of protein were loaded onto CriterionTM TGXTM precast gel, separated by electrophoresis, and then transferred to a nitrocellulose membrane (Bio-Rad Laboratories). After blocked 1 h at RT in PBST-5% non-fat milk, the membranes were incubated with primary antibodies at 4 °C overnight and then the corresponding secondary antibody at RT for 1 h. Images were captured and analyzed using the Odyssey Laser Fluorescence Detection System (LI-COR Biosciences). The band intensities were quantified by ImageStudioLiteVer 5.2 (LI-COR Biosciences). The primary antibodies were goat anti-NGAL (R&D Systems), rat anti-KIM-1 (R&D Systems), rabbit anti-BMPR1A (Santa Cruz Biotechnology), rabbit anti-αSMA (Abcam), mouse anti-glyceraldehyde 3-phosphate-dehydrogenase (Novus Biologicals), and rabbit anti-β-actin (Cell Signaling Technology).
6
Kidney Protein Profiling by IP-MS
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Immunoprecipitation was performed on kidney homogenate from aldosterone treated and control rats with the Santa Cruz H-95 Antibody (Table 1 ). First, the protein concentration in samples was measured using BCA protein assay reagent (Thermo Fisher Scientific). Then, volumes of kidney homogenate containing 350 µg protein were incubated with 40 µg of antibody in RIPA buffer containing 1% SDS, 0.05 M EDTA, 0.1 M Tris-HCl, 1% Na-dehydroxylate, 0.2 M NaCl, 1% Na Nonidet, and protease inhibitor. The mixture was incubated at 4°C for 1 hour. Subsequently, an equivalent volume of protein A beads were added to reaction mixtures and incubated with gentle rotation for 1 hour at room temperature. Then, samples were washed using RIPA buffer and eluted in sample buffer. Controls included omission of H-95 antibody and omission of beads. Samples were separated on 4–15% 26-well criterionTM TGXTM precast gel (BioRad), stained with Coomassie blue and lanes were excised manually before mass spectrometry.
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