Following treatment, H9c2 cells were scraped using a scraper and the protein lysates were extracted using a solubilization buffer (containing RIPA and 1% protease inhibitor). The debris was removed by centrifugation at 12,000 × g for 15 min at 4°C. The protein concentration was determined using a BCA assay kit. The protein samples (80 µg) were added to 5X loading buffer (Beyotime Institute of Biotechnology) and heated at 95°C for 5 min using metal heating block. Protein samples were separated using 10% SDS-PAGE and transferred onto a NC membrane, at a constant current of 300 mA for 1 h under wet transfer conditions. The NC membranes were blocked with 5% skimmed milk at room temperature, and the proteins were incubated with the following antibodies overnight at 4°C: NKA (1:200), c-Src (1:200), p-Src (1:1,000), Erk1/2 (1:1,000), p-Erk1/2 (1:1,000), Bax (1:500), Bcl-2 (1:500), caspase-3 (1:500), cleaved-caspase-3 (1:500) and GAPDH (1:1,000). The membranes were then incubated with the following fluorescent secondary antibodies: Goat anti-rabbit (1:10,000) and goat anti-mouse (1:10,000). The bands were captured and quantified using an Odyssey Imaging System (LI-COR Biosciences).
Loading buffer
Manufactured by Beyotime
Sourced in China, United States
Loading buffer is a solution used in various laboratory techniques, such as gel electrophoresis, to prepare samples for analysis. It is designed to increase the density of the sample, allowing it to sink into the gel matrix during the loading process. The buffer typically contains glycerol or other high-density compounds, as well as dyes that help track the progress of the separation.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (46 mentions)
Ripa lysis buffer, by Beyotime (42 mentions)
Ripa buffer, by Beyotime (36 mentions)
Bca kit, by Beyotime (27 mentions)
Bca protein assay kit, by Beyotime (24 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (16 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (13 mentions)
Gapdh, by Cell Signaling Technology (9 mentions)
Polyvinylidene fluoride membrane, by Merck Group (9 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (8 mentions)
Ripa buffer, by Thermo Fisher Scientific (8 mentions)
β actin, by Abcam (7 mentions)
Chemiluminescence imaging system, by Bio-Rad (7 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail tablet, by Roche (6 mentions)
Ecl kit, by Thermo Fisher Scientific (6 mentions)
Bca kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Lysis buffer, by Beyotime (5 mentions)
Goat anti rabbit igg, by ZSGB-BIO (5 mentions)
249 protocols using loading buffer
1
Western Blot Analysis of Protein Expression
2
Antioxidant and Metabolic Regulation in Cells
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EDTA, Tris, NaCl, HCl, NaOH, cetonitrile, trifluoroacetic acid, cytochrome C, aprotinin, bacitracin, glycine-glycine-tyrosine–arginine, and glycine-glycine-glycine were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Assay kits for biochemical detection of total antioxidant capacity (TAC), glutathione peroxidase (GPX), catalase (CAT), Malondialdehyde (MDA), BUN, LD, GN, LDH, Na+k+ATPase, Ca2+Mg2+ ATPase, and GOT were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). The bicinchoninic acid (BCA) assay kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). TRIzol reagent was purchased from Vazyme Biotech Co., Ltd. (Suzhou, China). RIPA lysis buffer and 5x loading buffer were purchased from Beyotime Institute of Biotechnology (Shanghai, China). Gene primers were designed and synthesized by Generay Biotech Co., Ltd. (Shanghai, China). Primary antibodies against GAPDH, p-AMPK, AMPK, SIRT1, PGC1α, TFAM, PPARσ, and PPARα were purchased from Abcam Co., Ltd. (Shanghai, China). Secondary antibody was supplied by LI-COR, Inc. (Lincoln, Nebraska, USA).
3
Protein Expression Analysis Protocol
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The treated cells were lysed in an appropriate volume of RIPA lysis buffer (Servicebio, China) containing 1% phenylmethanesulfonyl fluoride (Servicebio, China) and placed on ice for 30 min. Next, the mixture was centrifuged at 12,000 rpm for 15 min at 4°C. The supernatant was collected and protein concentration was determined by the bicinchoninic acid assay (Beyotime Biotechnology, Shanghai, China). Then, 5x loading buffer (Beyotime Biotechnology, Shanghai, China) was added to the supernatant and heated at 100°C for 15 min. The proteins were separated on sodium dodecyl sulfate-polyacrylamide gels (Beyotime Biotechnology, Shanghai, China) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore). The membrane was blocked in 5% skimmed milk at room temperature for 2 h and then incubated with primary antibody (β-actin antibody was diluted 1:4000, other antibodies (MMP-9, N-CAD, Twist, Vimentin and ZEB2) were diluted 1:1000) overnight at 4°C, followed by incubation with appropriate secondary antibody for 2 h at room temperature. Protein expression was detected using the ECL kit (Advansta, San Jose, CA, USA). The primary antibodies (β-actin, MMP-9, N-CAD, Twist, Vimentin and ZEB2) and the secondary antibody were purchased from Servicebio.
4
Western Blot Protocol for Protein Analysis
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For preparation of cell extracts, cells were washed thrice with ice-cold PBS, exposed to the recommended volume of RIPA lysis buffer (Beyotime, China) and placed on ice for 20 min. After centrifugation at 12000 rpm for 5 min at 4 °C, the supernatants were collected and diluted in 5X loading buffer (Beyotime, China). The samples were electrophoresed by SDS-PAGE and transferred to polyvinylidene fluoride membranes. After 2 hours of blocking with 5% fat free milk in TBST (TBS with 0.1% Tween-20) at room temperature, the membrane was incubated overnight with antibodies at 4 °C, washed three times with TBST, incubated for 2 h at room temperature with HRP-conjugated secondary antibodies, and then washed three times with TBST. Finally, immunoreactive protein was visualized with an enhanced chemiluminescence detection kit (Beyotime, China). The levels of target proteins were measured by densitometry.
5
Quantification of Mesenchymal Proteins by Western Blot
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The total NGM and GMAH mesenchymal proteins were mixed with 5X loading buffer (Beyotime Institute of Biotechnology) and boiled for 5 min. The proteins had been quantified using a bicinchonic acid protein assay kit (Beyotime Institute of Biotechnology). Next, the samples were separated using 10% gradient SDS-PAGE gels at 30 µg per lane and transferred onto PVDF membranes (Merck KGaA). The membranes were blotted with 5% fat-free milk suspended in TBST at room temperature for 1 h, incubated at 4°C overnight with S100 calcium-binding protein A6 (S100A6) antibody (1:1,000) (sc-53950; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and superoxide dismutase 3 (SOD3) antibody (1:1,000) (sc-58427; Santa Cruz Biotechnology, Inc.), washed and then incubated with goat anti-mouse IgG-HRP (1:2,000) (sc-2005; Santa Cruz Biotechnology, Inc.) at room temperature for 2 h. Detection of immunoreactivity was achieved using enhanced chemiluminescence (GE Healthcare Life Sciences).
6
Histone Modification Analysis by Western Blot
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Cells were washed with PBS after collection and lysed in SDS lysis solution supplied with protein inhibitor cocktail (Beyotime, P0013G) on ice for 5 min. Lysates were centrifuged at 12 000 rpm for 10 min at 4 °C. The supernatants were mixed with 5X loading buffer (Beyotime, P0015) and boiled at 95 °C for 15 min. Proteins were separated by SDS‐PAGE with 15% percentage gels, transferred to PVDF membranes (Bio‐Rad, 1620177) and blocked in 5% skim milk in TBST (20 mm Tris, 150 mm NaCl, and 0.1% Tween‐20) for 1 h. Membranes were incubated in TBST with primary antibodies (H3K27me3(CST, C36B11), H3K4me3(Abcam, ab213224), H3K27Ac (Abcam, ab177178) and Histone H3(Abcam, ab1791)) at 4 °C overnight. After washed with TBST 3 times, membranes were incubated in TBST with horseradish peroxidase (HRP) conjugated anti‐rabbit secondary antibody at room temperature for 1 h. After being washed with TBST 3 times, protein bands were visualized in Western Blot imaging system with ECL chemiluminescence kit (Vazyme, E422‐01) and quantified using ImageJ.
7
Western Blot Analysis of Lung Proteins
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Lung tissues or cells were lysed in lysis buffer. Lysates were separated from debris by centrifugation (4 °C, 12000 r/min) for 10 min and the supernatants were collected. Protein concentration was determined by bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Rockford, IL). Then the supernatants were boiled in 5X loading buffer (Beyotime Biotechnology, China) for 5 minutes. Samples were separated on 10% of polyacrylamide gels and transferred to polyvinylidene fluoride membranes. After blotting, the membranes were blocked in Tris-buffered saline with 0.05% Tween 20 (TBST) containing 5% nonfat dry milk for 90 min at room temperature, and then, incubated with TBST containing the specific primary antibodies overnight at 4 °C. The membranes were washed with TBST, and then incubated with HRP conjugated secondary antibodies in TBST. After washing, signals were revealed using the Pierce ECL Plus Western Blotting Substrate (Thermo Scientific, Rockford, IL). Western blots were scanned and densitometry ratios normalized to GAPDH content were analyzed using ImageJ software (NIH, US).
8
Western Blot Protein Expression Analysis
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Western blot assay was performed as previously described [10 (link)]. Briefly, protein was extracted with RIPA lysis buffer (Beyotime Biotechnology, China) on ice, quantified using the Pierce™ BCA Protein Assay Kit (Invitrogen, USA), and mixed with 5X loading buffer (Beyotime). The mixture was heated at 95 °C for 10 min to denature the protein and then loaded into the SDS-PAGE system. The primary antibodies used were anti-RUNX-2 rabbit mAb (Cell Signaling Technology, USA) and anti-GAPDH rabbit pAb (Goodhere Biotechnology, China), while the secondary antibody was goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, USA). The fluorescence of the cell membranes was scanned using an ImageQuant Las 4000 (Life Technologies, USA). To quantify the outcome, ImageJ software was used to analyse the bands, and each band was repeated three times.
9
Western Blot Protein Analysis Protocol
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Proteins were extracted from tissues or cells using RIPA lysis buffer (Beyotime). After centrifugation, the supernatant was mixed with 5X loading buffer (Beyotime) and boiled. The denatured proteins were separated by 4–12% or 4–20% SDS polyacrylamide gel (GenScript) and electrophoretically transferred to PVDF membranes (Millipore). The membranes were blocked with 5% nonfat dry milk for 1 h and probed with antibodies against TIPARP (1:100, Abcam ab170817 for Fig. 2 ; 1:500 ab84664 for the others), GAPDH (1:1000, Cell Signaling Technology #5174), β-actin (1:1000, Cell Signaling Technology #3700), collagen type I (1:1000, Proteintech No. 14695-1-AP), collagen type IV (1:1000, Proteintech No. 55131-1-AP), fibronectin (1:1000, Proteintech No. 15613-1-AP), and α-SMA (1:1000, Abcam ab7817). The membranes were then incubated with peroxidase-linked goat anti-mouse/rabbit IgG secondary antibody (1:3000, Yeasen). Signals were captured on a BioSpectrum imaging system (Ultra-Violet Products) or Kodak Molecular Imaging Software (Kodak).
10
Western Blot and RT-qPCR Analysis Protocol
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RIPA lysis buffer, BCA Protein Assay kit and 5X loading buffer were purchased from Beyotime Institute of Biotechnology. Rabbit polyclonal anti-FXR (cat. no. ab28676) and anti-TGR-5 (cat. no. ab72608) were purchased from Abcam. The mouse monoclonal antibody to β-actin (cat. no. sc517582) was purchased from Santa Cruz Biotechnology, Inc. The goat anti-Rabbit IgG (cat. no. BS13278) and goat anti-Mouse IgG (cat. no. BS12471) were purchased from Bioworld Technology, Inc. eECL Western Blot kit (cat. no. CW0049A) was purchased from CoWin Biosciences. RevertAidFirst Strand cDNA Synthesis kit was purchased from Thermo Fisher Scientific, Inc. SYBR Premix Ex Taq was purchased from Thermo Fisher Scientific, Inc. The primers were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). The fluorescence RT-qPCR kit was purchased from Takara Biotechnology, Co., Ltd. TRIzol® and the immunohistochemistry kit were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The 5% bovine serum was purchased from Wuhan Boster Biological Technology. Ltd.
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