One hundred milligrams of liver tissue per sample were homogenized in a commercial total protein extraction solution (Servicebio, Wuhan, China). Total protein lysates were fractionated by 10% SDS-PAGE (Servicebio, Wuhan, China) and electro-blotted onto a Polyvinylidene Fluoride membrane (Millipore, Massachusetts, USA). The primary antibodies used in this assay were FAS (1:1,000, 37 KD), SREBP-1(1:1,000, 57 KD), and β-Actin (1:1,000, 40 KD), which were purchased from Servicebio (Wuhan, China). Membranes were blocked with 5% non-fat milk for 1 h in Tris-Buffered Saline Tween buffer and probed with primary antibodies overnight at 4°C. Then, membranes were incubated with horseradish peroxidase-conjugated secondary antibody. Protein bands were visualized using chemiluminescence reagent (Millipore, Massachusetts, USA). The band values were calculated using AlphaEase FC software (Alpha Innotech).
Sds page
Manufactured by Wuhan Servicebio Technology
Sourced in China
SDS-PAGE is an analytical technique used to separate and analyze proteins based on their molecular weight. It utilizes an electric current to move charged proteins through a polyacrylamide gel, allowing for the separation and visualization of different protein components within a sample.
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Lab products found in correlation
Pvdf membrane, by Merck Group (5 mentions)
Ripa buffer, by Beyotime (3 mentions)
Pvdf membrane, by Wuhan Servicebio Technology (3 mentions)
Bca kit, by Wuhan Servicebio Technology (3 mentions)
Phenylmethanesulfonyl fluoride, by Beyotime (2 mentions)
Alphaeasefc software, by Bio-Techne (2 mentions)
Phosphatase inhibitor, by Wuhan Servicebio Technology (2 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Ripa lysis buffer, by Wuhan Servicebio Technology (2 mentions)
Ripa cleavage buffer, by Thermo Fisher Scientific (2 mentions)
Gb25301, by Wuhan Servicebio Technology (2 mentions)
Chemiluminescence reagent, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
β actin, by Wuhan Servicebio Technology (1 mentions)
Srebp1, by Wuhan Servicebio Technology (1 mentions)
Mpo antibody, by Abcam (1 mentions)
Bca protein concentration determination kit, by Wuhan Servicebio Technology (1 mentions)
Ecl kit, by Fdbio (1 mentions)
Gapdh antibody, by ZSGB-BIO (1 mentions)
20 protocols using sds page
1
Western Blot Analysis of Liver Proteins
2
Western Blot Protein Analysis
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Western blot analysis was performed between groups. Total proteins were extracted from cell samples. BCA protein concentration determination kit (Servicebio, Wuhan, China) was used to determine the total protein concentration. The proteins were separated by 10% SDS-PAGE (Servicebio, Wuhan, China) and transferred to cellulose nitrate membrane. The membrane was sealed in 5% degreased dry milk in 37°C TBS buffer (Servicebio, Wuhan, China) for 1 h and incubated overnight with primary antibodies (MPO antibodies, Abcam, Europe) at 4°C. Then, the membranes were washed three times in TBST and incubated with secondary antibodies at room temperature for 30 min. A scanner (EPSON V300, Japan) was used to sweep membrane and upload band data. Adobe PhotoShop (Adobe, USA) software was used to process the color, and Alpha Innotech (Alpha Innotech, USA) software was used to analyze the gray values of band. Relative expression level of protein = (gray value of the target protein band)/(gray value of the GAPHD protein band).
3
Protein expression analysis of renal cancer
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RIRA lysate buff was used to extract total protein from renal cancer cell lines (A498, 786-O, ACHN, Caki-1, and OSRC-2) and normal renal tubular epithelial cells (HK-2), and four pairs of ccRCC tissues and matched adjacent normal tissues from our study centre. Protein concentrations were determined by the BCA method using a bicinchoninic acid kit (CoWin Biosciences). Then, 30ug of total protein was separated by 10% SDS-PAGE (Servicebio, China) and transferred to PVDF membranes (0.45 mm,Immobilon-P Transfer Membrane). The membrane was subsequently blocked for 1 h at room temperature using 5% skim milk and inclubated with corresponding DBT antibody (Affinity Biosciences,DF13569,Rat, 1:1,000) or GAPDH antibody (ZSGB-BIO,TA-08,Mouse, 1:1,000) overnight at 4°C. After rinsing with TBST (Servicebio, China), the membrane was placed in the corresponding secondary antibodies and incubated for 1 h at room temperature, and rinsed again with TBST (Servicebio, China), and target protein expression levels were detected with an ECL kit (Fdbio science, China,Hangzhou) and a DNR Bioimaging System, analysed by using GraphPad Prism 9.
4
Quantifying Protein Levels in COL-4A1-Treated HUVECs
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WB was used to quantify protein levels in the samples. HUVECs were treated with COL-4A1 (0 μM, 30 μM) for 24 h. Proteins were extracted using RIPA buffer, PMSF (Beyotime Institute of Biotechnology, China) and phosphatase inhibitors (Servicebio, Wuhan). The protein samples were then separated by 10% SDS-PAGE (Servicebio, Wuhan) and transferred to nitrocellulose membranes. Then, the membranes were blocked with 5% skim milk (Guangzhou Saiguo Biotech Co., LTD) for 2 h and incubated with primary antibodies against TGF-β (Abcam, 1:1000, ab179695), PI3K (Abcam, 1:500, ab32569), AKT (CST, 1:1000, #9272), p-AKT (CST, 1:1000, #9271), and β-actin (Proteintech, 1:1000, 66009-1-lg) overnight at 4 °C. On the following day, the membranes were washed 3 times for 10 min/wash and incubated with secondary antibodies (1:5000). Finally, the membranes were washed 3 times for 10 min/wash and exposed to FluorChem M from ProteinSimple (GeneTech Biotechnology, Shanghai).
5
Protein Expression Analysis by Western Blot
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Cells and tissues were collected and lysed with RIPA buffer containing phenylmethanesulfonylfluoride (Beyotime, China). Equivalent amounts of total protein were separated by 10% SDS-PAGE (Servicebio, China), then transferred to PVDF membranes (Servicebio, China). The membranes were then blocked in TBST containing 5% nonfat milk, and the membranes were incubated with primary antibodies overnight at 4 °C. The membranes were then incubated with secondary antibodies. An ECL kit (Multisciences, China) was used to detect proteins. Primary antibodies against FOXO1 (2880) and cyclin D1 (2978S) were purchased from Cell Signaling Technology. Antibodies against p21 (ab109520) and GAPDH (ab9485) were purchased from Abcam.
6
Western Blot Analysis of Protein Expression
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Cells were collected and lysed with RIPA buffer containing phenylmethanesulfonyl-fluoride (Beyotime, China). Equivalent amounts of total protein were separated by 10% SDS-PAGE (Servicebio, China) and transferred to the PVDF membrane (Servicebio). Then, the membranes were blocked in TBST containing 5% nonfat milk and probed with primary antibodies at 4 °C overnight, followed by incubation with secondary antibodies at room temperature for 1 h. Finally, the immunoreactive bands were developed and examined on the gel imager. The primary antibodies were as follows: RhoBTB3 (AF9183), HIF1A (AF1009), GGT1 (DF6610), VEGFA (AF131), andβ-actin (AF7018) from Affinity.
7
Western Blotting of Colon Proteins
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The total proteins were extracted from human colon tissues and cultured cells for western blotting analysis, mixed with 1× cell lysis buffer containing protease inhibitors (Beyotime, Wuhan, China). Equal amount of denatured proteins separated with 10% SDS-PAGE (Servicebio, Wuhan, China), were transferred to polyvinylidene fluoride (PVDF) membranes (Servicebio). The PVDF membranes with the target proteins were blocked with 5% skimmed milk (Servicebio) and incubated with various primary antibodies (1:1000, Proteintech, Wuhan, China) at 4 °C overnight. After rinsing, the PVDF membranes were further incubated with the appropriate secondary antibody (Servicebio) for 1 h at room temperature. An ECL reagent (Servicebio) was employed to visualized band signals, and the band densities were detected and quantified by using the ImageJ software.
8
Western Blot Analysis of EGFR and AKT
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Cells were lysed using RIPA buffer supplemented with protease and phosphatase inhibitors (TargetMol, America), and the protein supernatant was stored at −80°C. The protein concentration was detected by the BCA (Beyotime, China) method. After reduction by heating at 99°C for 20 min, the sample proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Servicebio, China) and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 90 min followed by incubation with the primary antibody at 4°C overnight and then with the secondary antibody for 2 h at room temperature. Finally, enhanced chemiluminescence (ECL) reagent (EpiZyme, China) was applied to visualize the protein bands. The primary antibodies involved in this work include EGFR (1:1000, CST, America), p-EGFR (1:1000, CST, America), AKT (1:1000, CST, America), p-AKT (1:1000, CST, America), and GAPDH (1:1000, Abclonal, China). Secondary antibodies were purchased from ABclonal, China.
9
Quantification of Protein Levels in HTR-8/SVneo Cells
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WB was used to quantify the protein levels in samples. HTR‐8/SVneo cells were transfected with LINC00534 OE plasmids or siRNAs for 48 h. A RIPA buffer supplemented with PMSF (Beyotime Institute of Biotechnology) and phosphatase inhibitors (Servicebio) was used to extract proteins. After separation by 10% SDS‐PAGE (Servicebio), the samples were transferred to nitrocellulose membranes. Further, after blocking with 5% skim milk (Guangzhou Saiguo Biotech Co., LTD) for 2 h, membranes were incubated with primary antibodies against PI3K (p110β) (CST, 1:1000, #3011), ERK1 (Abcam, 1:1000, ab32537), NF–κB p65 (CST, 1:1000, #8242), STAT3 (Abcam, 1:2000, ab68153), PTEN (Abcam, 1:5000, ab32199), and β‐ACTIN (Proteintech, 1:1000, 66009‐1‐lg) overnight at 4°C. The membranes were washed three times for 10 min each and incubated with secondary antibodies the following day.
10
Quantitative Protein Analysis of Podocytes
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Protein exacted from podocytes were prepared in RIPA lysis buffer (Boster Biological Technology, Wuhan, Hubei, China), and were estimated using BCA protein assay kit (Beyotime). Proteins were separated by SDS-PAGE (G2003, Servicebio), transferred onto PDVF membranes (Millipore) after electrophoresis, and blocked by 5% skimmed milk at room temperature for 1 h. After incubation with primary antibodies at 4 °C overnight, the membranes were incubated with secondary antibodies. We quantified the band area using AlphaEase FC software (Alpha Innotech) to reflect the relative expression of target proteins. The results were corrected to the band intensity of GAPDH. The antibodies used are shown in Table 1 .
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