The tissues or cells were lysed to obtain protein samples using lysis buffer mixed with RIPA (Beyotime Institute of Biotechnology, Shanghai, China), PMSF (Wuhan Boster Biological Technology, Ltd.), and cocktail protease inhibitors (Roche Diagnostics, Indianapolis, IN, USA). 30 μg protein sample was added to 10% SDS-PAGE and separated at a stable voltage of 90 V. Then, the protein was transferred into polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA) at a stable current of 250 mA for 105 min. After being blocked in 5% milk for 1.5 hat room temperature, the transferred PVDF membranes were incubated with primary antibodies against SLC17A9 (1:1000; Proteintech, USA), Snail (1:1000, ABclonal), E-cadherin (1:1000, ABclonal), vimentin (1:1000, ABclonal), and β-actin (1:1000, Proteintech) at 4 °C overnight. Next, the membranes were incubated using secondary antibodies (1:10,000; BA1020; Wuhan Boster Biological Technology Ltd.) at room temperature for 2 h. Lastly, ChemiDoc-XRS+ (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used for the detection of protein levels.
Phenyl methyl sulfonyl fluoride (pmsf)
Manufactured by Wuhan Boster Biological Technology
Sourced in China, United States
PMSF is a protease inhibitor commonly used in molecular biology and biochemistry applications. It functions by irreversibly inhibiting serine proteases, preventing the degradation of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Roche (6 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Ba1020, by Wuhan Boster Biological Technology (5 mentions)
Bm3876, by Wuhan Boster Biological Technology (4 mentions)
Chemidoc xr, by Bio-Rad (3 mentions)
Bicinchoninic acid kit, by Beyotime (2 mentions)
Snail, by ABclonal (1 mentions)
β actin, by Proteintech (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Vimentin, by ABclonal (1 mentions)
E cadherin, by ABclonal (1 mentions)
Ab205394, by Abcam (1 mentions)
Radioimmunoprecipitation assay buffer, by Wuhan Boster Biological Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab8226, by Abcam (1 mentions)
Gb23303, by Wuhan Servicebio Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Ripa buffer, by Wuhan Boster Biological Technology (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
7 protocols using phenyl methyl sulfonyl fluoride (pmsf)
1
Western Blot Analysis of Protein Markers
2
Protein Quantification and Western Blot
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Tissues and cells are pyrolyzed in a protein lysis system containing RIPA, PMSF (Wuhan Boster Biological Technology, Ltd, Wuhan, China), and protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Protein concentration was measured at 562 nm. A total of 30 μg of proteins were presented in SDS-PAGE gel system then separated and transferred to polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Bedford, MA, USA) for 90 min. After transfer to the PVDF membrane, the membrane was blocked in PBS containing 5% skim milk for 1 h and then incubated with antibodies against BMP1 (1:1000; ab205394; Abcam Co., Ltd) and GAPDH (1:2000; BM3876; Wuhan Boster Biological Technology, Ltd, Wuhan, China) at 4 °C overnight. The next day the membranes were washed and incubated with secondary antibodies (1:5000; BA1020; Wuhan Boster Biological Technology, Ltd, Wuhan, China) at room temperature (25 °C) for 2 h. Finally, the membranes were washed and detected by Biosense SC8108 Gel Documentation System with GeneScope V1.73 software (Shanghai BioTech, Shanghai, China).
3
Quantitative Protein Expression Analysis
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Tissues and cells were pyrolysed in a protein lysis system containing radioimmunoprecipitation assay buffer (Wuhan Boster Biological Technology, Ltd.), a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) and PMSF (Wuhan Boster Biological Technology, Ltd.). Protein concentrations were measured with a bicinchoninic acid kit (Beyotime Institute of Biotechnology) at 562 nm. Thirty micrograms of each protein were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (EMD Millipore, Bedford, MA, USA) for 90 min. After the transfer, the membranes were blocked in phosphate-buffered saline with 5% non-fat milk for one hour and then incubated with antibodies against SLC4A4 (1:1000; A5332; ABclonal Biotech Co., Ltd.), KRAS (1:1000; A11059; ABclonal Biotech Co., Ltd.) and GAPDH (1:2000; BM3876; Wuhan Boster Biological Technology, Ltd.) at 4°C overnight. The next day, the membranes were washed and then incubated with secondary antibodies (1:5000; BA1020; Wuhan Boster Biological Technology, Ltd.) at room temperature (25°C) for two hours. Finally, the membranes were washed, and protein levels were detected with a ChemiDoc-XRS+ (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
4
Western Blot Analysis of ORC6 Protein
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We followed the methods of Qiufeng Pan et al. 202016 (link). Cells and tissues were lysed in a protein lysis system including PMSF (Wuhan Boster Biological Technology, Ltd, Wuhan, China), protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA), and RIPA buffer (Beyotime Institute of Biotechnology). Protein concentration was measured by the BCA kit (Beyotime Institute of Biotechnology). A total of 30μg proteins were presented and separated in 10% SDS-PAGE. Then, the gel system was transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Bedford, MA, USA) for 90 mins at 90 V, after which the PVDF membrane was blocked in 5% nonfat milk dissolved in PBS for 1 h and then incubated with antibodies against ORC6 (1:1,000; A5426; Abclonal Biotec Co., Ltd) and β-actin (1:3,000; ab8226; Abcam Co., Ltd) at 4°C overnight. The next day, the membrane was washed and incubated with secondary antibodies (1:3,000; GB23303; Servicebio, Inc.) for 2 h at room temperature. Finally, the proteins were visualized with ChemiDoc-XRS+ (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
5
Western Blot Analysis of PLP2 Protein
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Tissues and cells were obtained by protein lysis with RIPA buffer (Wuhan Boster Biological Technology, Ltd., Wuhan, China), PMSF (Wuhan Boster Biological Technology, Ltd., Wuhan, China) and a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Protein concentrations were measured with a bicinchoninic acid kit (Beyotime Institute of Biotechnology). Western blotting was performed according to the manufacturer's instructions with polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Bedford, MA, USA); proteins were blocked in PBS with 5% non-fat milk and incubated with antibodies against PLP2 (1:1000; A06255; Boster Biological Technology, Ltd., USA) and GAPDH (1:2000; BM3876; Wuhan Boster Biological Technology, Ltd., Wuhan, China) overnight. After 14 h, membranes were incubated with secondary antibodies (1:5000; BA1020; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 2 h, and the signal was detected by the ChemiDoc XRS+ system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
6
Protein Extraction and Western Blot Analysis
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Cells are pyrolyzed in RIPA, protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) and PMSF (Wuhan Boster Biological Technology, Ltd., Wuhan, China) protein lysis system. A total of 30 μg proteins were added to the SDS-PAGE gel system, then separated the proteins and transferred them to polyvinylidene fluoride (PVDF) membrane (EMD Millipore, Bedford, MA, USA) within 90 mins. After protein transfer to the PVDF membrane, the membrane was blocked in 5% skim milk within 1 hr. After cleaning the membrane with PBS 3 times, incubated the membrane with antibody against GAPDH (1:2000; BM3876; Wuhan Boster Biological Technology, Ltd., Wuhan, China) or IREB2 (1:1000; 23829-1-AP; Proteintech, Rosemont, USA) at 4°C overnight. After incubated the membranes 12–16 hrs, the membranes were washed and incubated the membranes for 2 h at room temperature with secondary antibodies (1:5000; BA1020; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Finally, the membranes were detected by Biosense SC8108 Gel Documentation System with GeneScope V1.73 software (Shanghai BioTech, Shanghai, China) as previous research.23 (link)
7
Western Blot Analysis of Protein Expression
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After 48 h transfection, cells were lysed by RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with protease inhibitor PMSF (Wuhan Boster Biological Technology, Ltd, Wuhan, China). Protein concentration was detected with bicinchoninic acid (BCA) method. Separated proteins were denatured at 95 °C for 5 min. Equal amount of protein (20 μg) was added into each well in 12% SDSPAGE, and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). Afterwards, PVDF membranes were blocked in 5% skim milk for 1 h at room temperature, following the incubation with specific primary antibodies at 4 °C overnight. PVDF membranes were then sealed with secondary antibody for 1 h at 37 °C. The protein signals were visualized with ECL solution (Millipore) and scanned by QUANTITY ONE software (Bio-Rad, Hercules, CA, USA). All antibodies used in this study were listed as the following: anti-SPOP (Abcam, ab192233); anti-CHAF1A (Cell signaling Technology, CST#5480); anti-HA (Abcam, ab9110); anti-p62 (Cell signaling Technology, CST#23,214); anti-Beclin-1 (Cell signaling Technology, CST#4122); anti-β-actin (Cell signaling Technology, CST#41,470); anti-FLAG (Cell signaling Technology, CST#14,793).
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