Cultured cells or tissues were prepared with the T-PER tissue protein extraction reagent (2% SDS and 60 mM Tris HCl, pH 6.8) with the cocktail of proteinase inhibitors (Roche, Indianapolis, IN, USA) in it. The total protein tissue or cultured cells were homogenized in lysis buffer and were loaded onto the gel (20–40 μg) for electrophoresis. Proteins then were transferred onto 10% SDS-PAGE (sodium dodecylsulfate polyacrylamide gel electrophoresis) and were then transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). The electroblotted membranes were blocked by TBS containing 5% non-fat milk (Santa Cruz, Dallas, TX, USA) and were probed with primary antibodies overnight at 4°C and immunoblotted with specific antibodies. Primary antibodies against the following proteins were used: UCP1, PGC1α, PRDM16, Cidea (Abcam, Cambridge, UK), PPARγ (CST, Danvers, MA, USA), 422/Ap2 (Santa Cruz Biotechnology), RPS3A (ProteinTech Group, Rosemont, IL, USA), Cyto c (CST), Actin (Sigma, St. Louis, USA).
Cocktail of proteinase inhibitors
Manufactured by Roche
Sourced in Switzerland
The Cocktail of Proteinase Inhibitors is a laboratory product designed to inhibit the activity of various proteolytic enzymes, known as proteinases. This product is a mixture of several different inhibitors that target a range of proteinase types, providing a broad-spectrum approach to protecting proteins from unwanted degradation during experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Cocktail of phosphatase inhibitors, by Roche (5 mentions)
Protein extraction reagent, by Thermo Fisher Scientific (4 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Immobilob western chemiluminescent hrp substrate, by Merck Group (2 mentions)
Cidea, by Abcam (1 mentions)
Rps3a, by Proteintech (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Semi dry apparatus, by Bio-Rad (1 mentions)
Mmp 13, by Santa Cruz Biotechnology (1 mentions)
Anti clu, by Santa Cruz Biotechnology (1 mentions)
Eif3i, by Abcam (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Qiagen (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Gapdh antibody, by Cell Signaling Technology (1 mentions)
Hsp70, by Proteintech (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
9 protocols using cocktail of proteinase inhibitors
1
Protein Expression Analysis in Cultured Cells
2
Protein Extraction and Western Blotting
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Protein extraction and western blotting were performed as previously described[14 (link)]. Cell lysates were extracted with the T-PER tissue protein extraction reagent (Pierce, Rockford, IL) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science). Equal amounts of total proteins (20 μg) were separated by 10% SDS-PAGE and transferred onto PVDF membrane using a Bio-Rad SemiDry apparatus. After blocking for nonspecific binding, the membranes were incubated with anti-CLU (1:200 dilution; Santa Cruz Biotechnology), MMP13 (1:200 dilution; Santa Cruz Biotechnology), p-Akt (1:1000 dilution; Cell Signaling Technology), Akt (1:1000 dilution; Cell Signaling Technology), EIF3I (1:800 dilution; Abcam, Hong Kong, China) or GAPDH (1:5000 dilution; Kang-Chen, Shanghai, China) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 h at room temperature. After washing three times in TBST, protein bands were visualized using chemiluminescence detection.
3
Western Blot Analysis of NPC2 and NPC1L1
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Western blot was performed according to previous study 15 . Briefly, tissue samples were homogenated in a RIPA buffer (Qiagen, China) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science). The protein concentrations were determined using the Bicinchoninic Acid (BCA) Kit (Pierce). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA). The membranes were incubated with primary NPC2 antibodies (A5413; Abclonal, USA) at 1:1500, or NPC1L1-HRP (ab201773; Abcam, USA): 1:2000) overnight at 4 °C. After washing the membrane, NPC1L1 was visualised using ECL development solution. For detection of NPC2, secondary antibodies incubated at room temperature for 1~2 h and NPC2 was visualised using ECL development solution.
4
Western Blot Analysis of Melanocyte Proteins
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The MCs were seeded in the 60 mm cell culture dish and allowed to grow to 60%-80%, and were stimulated by α-MSH and/or siRNA for 48 h. Then, the total protein of cells was extracted using RIPA lysis buffer with a cocktail of proteinase inhibitors (Roche, Mannheim, Germany) and a cocktail of phosphatase inhibitors (Roche, Mannheim, Germany) according to its protocol. 30 μg total protein was separated by 10% SDS-PAGE and transferred onto a PVDF membrane. After blocking by 0.1% BSA, the membranes were incubated with primary antibody TYR (#ab180753, Abcam, Cambridge, UK) and Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH, AP0066, Bioworld Technology, Inc., Minnesota, USA) overnight at 4° C and followed by an incubation period of 1 h at room temperature with secondary antibody (1:10000, LI-COR Biosciences, USA). Bands were detected by the enhanced LI-COR Odyssey infrared imaging system (LI-COR Biosciences, NE, USA).
5
Western Blot Analysis of Protein Expression
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Briefly, total protein of tissue samples and cell lines was extracted using protein extraction reagent (Thermo Scientific) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science) according to its protocol. Equal amount of total protein (20 μg) was separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking for nonspecific binding, the membranes were incubated with following primary antibodies: E-cadherin (Abcam, 1:1,000), Vimentin (Abcam, 1:1,000), N-cadherin (Cell Signaling Technology, 1:1,000), FMN2 (Abcam, 1:1,000), TSG101 (1:1,000, Proteintech, Chicago, USA), HSP70 (1:2,000, Proteintech, Chicago, IL, USA), and GAPDH antibody (1:2,000; Cell Signaling Technology) overnight at 4°C. Then PVDF membranes were incubated with the appropriate secondary antibody (Santa Cruz Biotechnology, 1:10,000) for 1 h at room temperature. Bands were detected by a Bio-rad ChemiDoc XRS system.
6
Western Blot Analysis of LUSC Proteins
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Briefly, total protein of LUSC tissue samples and cell lines was extracted using protein extraction reagent (Thermo Scientific) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) and a cocktail of phosphatase inhibitors (Roche Applied Science) according to its protocol. Equal amount of total protein (20 μg) was separated by 10% SDS–PAGE and transferred onto a PVDF membrane. After blocking for nonspecific binding, the membranes were incubated with antibody FOXM1 (1:3000 dilution; proteintech; 13147-1-AP), CENPA (1:1000 dilution; Abcam; ab45694), CENPB (1:1000 dilution; Abcam; ab25734), CCNB1 (1:1000 dilution, Cell Signaling Technology; #4134), or β-actin (1:10000 dilution; Sigma; A2228) overnight at 4 °C and followed by an incubation period of 1 h at room temperature with secondary antibody (1:4000, Bioword; 20330016-1). Bands were detected by a Bio-rad ChemiDoc XRS system. Full scans of the western blots shown in Figs. 4 e, f, 5 g and 6a, b and Supplementary Figs. 5 c and 6c are provided in Source Data file.
7
Immunoblotting Analysis of HCV Proteins
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The HCV-transfected Huh 7.5 cells were lysed in a radioimmuno-precipitation assay buffer (50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 1% Nonidet P40, 0.5% sodium deoxycholate) containing a cocktail of proteinase inhibitors (Roche). The total protein for each sample was measured with a standard protein assay (Bio-Rad). Twenty-five micrograms of total protein for each sample was analyzed by 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked by incubating them with 5% skim milk. HCV proteins were detected with monoclonal antibodies specific to NS5A, horseradish peroxidase-conjugated goat anti-mouse immunoglobulin G (Bio-Rad) and a chemiluminescence substrate (Pierce). β-Actin was used as a control and was detected with an anti-β-actin monoclonal antibody (Sigma).
8
Protein Extraction and Western Blot Analysis
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The protein extraction reagent (Thermo Scientific) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) was used to isolate the total protein from cells or tissue samples. Equal amount of total protein was separated by 10% SDS-PAGE and transferred onto a PVDF membrane. Then, the membranes were blocked with 5% skimmed milk and incubated the membranes with primary antibodies at 4°C overnight and then incubated with secondary antibodies at room temperature for 2 h. The bands were examined by Immobilob™ Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA). The primary
9
Protein Extraction and Western Blot Analysis
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The protein extraction reagent (Thermo Scientific) with a cocktail of proteinase inhibitors (Roche Applied Science, Switzerland) was used to isolate the total protein from cells or tissue samples. Equal amount of total protein was separated by 10% SDS-PAGE and transferred onto a PVDF membrane. Then, the membranes were blocked with 5% skimmed milk powder and incubated the membranes with primary antibodies at 4 °C overnight and then incubated with secondary antibodies at room temperature for 2 h. The bands were examined by Immobilob™ Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA). The primary antibody and secondary antibodies were purchased from Cell Signaling Technology, and the detailed information list below: KEAP1(KEAP1
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