Total protein was extracted with a cell lysis reagent and the concentration was measured using a BCA protein assay kit (Beyotime Biotec, China). Protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred onto a polyvinylidene difluoride membrane (Millipore, MA, USA), which was then blocked with 5% skim milk powder dissolved in Tris-buffered saline-Tween. After incubation at room temperature for 1 h, the membrane was incubated overnight with primary antibodies at 4°C. The primary antibodies of anti-PPARG, anti-E-cadherin, anti-Vimentin, and anti-GAPDH were purchased from Abcam (MA, USA); anti-Snail from Gene Tex (CA, USA) and NF-κB from Cell Signaling Technology (MA, USA). The membranes were then incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (Abcam, MA, USA) at room temperature. The proteins were detected using an ECL detection Kit (Millipore, MA, USA). The signal density of protein bands was quantified using the Tanon-5200 Imaging System (Tanon Science & Technology, China). GAPDH was used as a loading control.
Tanon 5200 imaging system
Manufactured by Shanghai Tanon Science & Technology
Sourced in China
The Tanon 5200 Imaging system is a laboratory equipment designed for imaging and analyzing biological samples. It is capable of capturing high-quality digital images of gels, blots, and other specimens. The system utilizes advanced imaging technologies to provide accurate and reliable results.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Anti pparg, by Abcam (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Anti snail, by GeneTex (1 mentions)
Ecl detection kit, by Merck Group (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Ecl solution, by Vazyme (1 mentions)
Anti claudin 1 antibody, by Abcam (1 mentions)
Anti claudin 3 antibody, by Abcam (1 mentions)
0.2 μm polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab113509, by Abcam (1 mentions)
Ab237704, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab8227, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Proteintech (1 mentions)
8 protocols using tanon 5200 imaging system
1
Western Blot Analysis of Protein Expression
2
Protein Expression Analysis in A549 Cells
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After drug intervention or no drug intervention, A549 or A549/DDP cells were lysed and subjected to western blot analysis as a general method [16 (link)]. After treatment, the blots were probed with antibodies against Beclin 1, LC3, p62, cleaved caspase-3, p53, Bax, Bcl-2, FOXO3a, p-FOXO3a Ser253, p-FOXO3a Ser315, and p-FOXO3a Thr32; then, we detected the protein bands with ECL solution (Vazyme, Nanjing, China) using a Tanon 5200 imaging system (Tanon Science & Technology, Shanghai China) [17 (link)].
3
Colon Tissue Protein Analysis
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Proteins extracted from the colon tissue were separated by 8–12% SDS-PAGE after the concentration was detected by the BCA protein assay kit (CW0014S, CoWin Biotech Co., Inc., Beijing, China). They were then transferred to 0.2 μm polyvinylidene fluoride membranes (Merck KGaA Co., Ltd., Darmstadt, Germany). Membranes were blocked with 5% skim milk for 1.5 h. After blocking, the membranes were incubated with anti-claudin 1 antibody (1/3000, Abcam Co., Inc., Cambridge, UK) and anti-claudin 3 antibody (1/1000, Abcam Co., Inc., Cambridge, UK) at 4 °C overnight. The membranes were washed with Tris-buffered saline Tween (TBST) and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG, or goat anti-rabbit IgG (CoWin Biotech Co., Inc., Beijing, China) for 1.5 h. The membranes were imaged with a Tanon 5200 imaging system (Tanon Science & Technology Co., Ltd., Shanghai, China).
4
Protein Extraction and Quantification from HepG2 and Huh7 Cells
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To extract total protein, both HepG2 and Huh7 cells were lysed with RIPA Lysis Buffer (Beyotime Institute of Biotechnology) as described previously (25 (link)). The lysate was centrifuged at 1,200 × g at 4°C for 10 min. The protein amount in the supernatant was quantified using a BCA assay. Subsequently, protein specimens (40 µg/lane) were added onto SDS-PAGE, and electrophoresis at 60 V. Proteins were then transferred to a polyvinylidene difluoride membrane (EMD Millipore). After blocking with 5% (w/v) non-fat dry milk at 37°C for 1 h, the membranes were incubated with primary antibodies against β-actin (1:2,000 dilution; cat. no. ab8226) and hnRNPA2B1 (1:1,000 dilution; cat. no. ab31645) at 4°C overnight. All antibodies were purchased from Abcam. Next, HRP-conjugated secondary antibodies (1:5,000 dilution; cat. no. HRP-60008, ProteinTech Group, Inc.) were added and incubated for 1 h at room temperature. The protein bands were detected using chemiluminescence (Pierce; Thermo Fisher Scientific, Inc.) and visualized on a Tanon 5200 Imaging system (Tanon Science & Technology Co., Ltd.).
5
Quantification of Cellular Proteins
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Total protein was extracted by cell lysis using RIPA Lysis Buffer (Beyotime Institute of Biotechnology, China). The lysate was centrifuged at 12,000 rpm at 4˚C for 10 min, and the supernatant was transferred to a new tube to quantify total protein using the BCA assay. Subsequently, electrophoresis was conducted with 12% SDS gels followed by transfer onto PVDF membranes (EMD Millipore). Following blocking with 5% (w/v) non-fat dry milk, the membranes were incubated with primary antibodies against β-actin (ab8227, 1:1,000), SLC1A5 (ab237704, 1:1,000), aspartate aminotransferase (GOT1; ab239487, 1:1,000 dilution), and glutaminase 2 (GLS2; ab113509, 1:1,000) and all antibodies were purchased from Abcam. Subsequently, the appropriate HRP-conjugated secondary antibodies (1:5,000; ProteinTech Group) were applied. The protein bands were detected using a chemiluminescence-based method (Pierce; Thermo Fisher Scientific, USA) on a Tanon 5200 Imaging system (Tanon Science & Technology Co., China). The expression levels of the proteins in each sample were normalized to those of β-actin.
6
Western Blot Analysis of Liver Proteins
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Liver tissues were homogenized using a Tissue Protein Extraction Reagent (Beyotime, Shanghai, China) for 30 min at 4°C. The homogenates were centrifuged at 12,000 g for 15 min at 4°C, and the protein concentration was determined using a Bicinchoninic Acid (BCA) Protein Assay Kit (Beyotime, Shanghai, China). Equal amounts of protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto nitrocellulose (NC) membranes. After blocking with 5% nonfat milk in TBST for 2 h at room temperature, the membranes were incubated overnight with anti-P53, anti-Bcl-2 (Wanlei Biology, Shenyang, China), anti-Bax, and anti-β-actin (Sangon Biotech, Shanghai, China) primary antibodies. The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibody (ImmunoWay, Plano, USA) at room temperature for 2 h. Following another wash with TBST, the blots were developed using a Meilunbio® fg super-sensitive ECL luminescence reagent (Meilunbio, Dalian, China) and imaged using the Tanon 5200 Imaging System (Tanon Science & Technology Co., Shanghai, China). The relative density of the target bands was quantified using ImageJ software.
7
Profiling HDAC6 Splice Variants
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Total RNA from HCT116 cells treated with or without compound 6d for 4 h and 8 h was extracted using Beyozol (R0011, Beyotime) according to the manufacturer’s instructions. The genomic sequence of HDAC6 spanning exons 26 to 29 was amplified using the forward primer 5′-GGGGGATCCGGGGCCTCAGAATCTCAG-3′ and the reverse primer 5′-GGGGCTCGAGAACAGCTTGTACTTTATT-3′. RT-PCR was performed using BeyoFast™ SYBR Green One-Step qRT-PCR Kit (D7268S, Beyotime) according to manufacturer’s instructions, and then the PCR products were assessed to detect the relative abundance of different splice forms by applying to 2% agarose gels. The results were analyzed using the Tanon 5200 Imaging System (Tanon Science & Technology Co., Ltd., Shanghai, China).
8
Western Blot Analysis of EMT Markers
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OC cell lysates were harvested using RIPA lysis buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.). The protein concentrations were determined using a bicinchoninic acid kit (cat. no. P0012, Beyotime Institute of Biotechnology) and 20 µg proteins were loaded in to each well. Proteins were separated using 10% SDS-PAGE and transferred to PVDF membranes (cat. no. IPVH00010; Merck KGaA). The membranes were blocked with 10% milk at room temperature for 1 h. Cells were blotted with specific primary antibodies against E-cadherin (1:3,000; cat. no. ab15148; Abcam), N-cadherin (1:3,000; cat. no. ab18203; Abcam), vimentin (1:3,000; cat. no. ab11256; Abcam), TJP3 (1:2,000; cat. no. 710857; Thermo Fisher Scientific, Inc.) or GAPDH (1:3,000; cat. no. ab9485; Abcam) at 4°C overnight and then incubated with a horseradish peroxide-conjugated goat anti-rabbit IgG H&L secondary antibody (1:10,000; cat. no. ab205718; Abcam) for 1 h at room temperature. The signal of proteins was visualized using ECL detection reagent (cat. no. 36208ES60, Shanghai Yeasen Biotechnology Co., Ltd.) with a Tanon-5200 imaging system (Tanon Science & Technology Co., Ltd.). ImageJ 1.8.0 software (National Institutes of Health) was used to quantify the western blots.
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