Dulbecco's modi ed Eagle medium (DMEM) culture medium and fetal bovine serum (FBS) were purchased from Hyclone (Logan, Utah); Hsa-miR-20a-5p mimic (GMUL0163595) from Shanghai GeneChem Chemical Technology, Co., Ltd., China; Cell counting kit-8 (CCK-8) from Dongren Chemical Technology Co., Ltd., Shanghai, China; RIPA buffer (#P0013B), phenylmethanesulfonyl uoride (PMSF, #ST506), and phosphatase inhibitor cocktail (#P1081) from Beyotime Biotechnology, Nantong, China; Bicinchoninic acid (BCA) protein assay kit (#23225) from Thermo Scienti c, Rockford, IL, USA; Trizol reagent from Invitrogen, USA; A cDNA synthesis kit (#RR037A) from Dalian Takara, China; Rabbit anti-PAR-1 antibody (#SAB4500823, 1:1000) from Sigma-Aldrich Company, Shanghai, China; Rabbit anti-GAPDH antibody (#AC001, 1:2000) from ABclonal Biotechnology Co., Ltd., HK; Goat anti rabbit IgG (H+L) secondary antibody (#V926-32211, 1:1000) from Li-Cor, Inc., Lincoln, NE.
Phenylmethanesulfonyl uoride
Manufactured by Beyotime
Sourced in China
Phenylmethanesulfonyl fluoride (PMSF) is a lab equipment product that functions as a serine protease inhibitor. It is commonly used in biochemical research and assays to prevent the degradation of proteins by proteolytic enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Beyotime (2 mentions)
Phosphatase inhibitor cocktail, by Beyotime (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rabbit anti gapdh antibody, by ABclonal (1 mentions)
Phosstop, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Bace1, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
3 protocols using phenylmethanesulfonyl uoride
1
Hsa-miR-20a-5p Modulates Cell Proliferation
2
Protein Extraction and Immunoblotting Protocol
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The total proteins were extracted from AAFs using a lysis buffer containing RIPA buffer (Beyotime, China), 10% PhosSTOP (Thermo), and 1 mM phenylmethanesulfonyl uoride (Beyotime). Next, 10% and 12% SDS-PAGE were used to separate the proteins, and then the proteins were transferred onto a polyvinylidene di uoride (PVDF) membrane (Millipore). The PVDF membrane was blocked in TBS/Tween with 5% BSA and subsequently incubated with the following primary antibodies: p-JAK1, JAK1, p-JAK2,
3
Protein Expression Analysis in Cells
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Cells or neurons were lysed in RIPA Lysis Buffer (Beyotime, Shanghai) which contained 1mM of Phenylmethanesulfonyl uoride (Beyotime, Shanghai) according to manufacturer's instructions. Cell lysate samples were electrophoresed on SDS-polyacrylamide gels in Tris-glycine buffer containing SDS.
Proteins were transferred to 0.22μm NC-membranes. Then membranes were blocked in 3% Bovine Serum Albumin (BSA) diluted in TBST buffer at room temperature, and were incubated overnight at 4℃ with primary antibodies (BACE1 [Abcam, UK, 1:2000], β-actin [Cell Signaling Technology, USA, 1:2000], APP [Thermo Fisher, USA, 1:500]). The membranes were then washed in TBST and incubated with secondary antibody for 1 hour at room temperature. The immunoreactivity of the proteins was detected using Chemiluminescent Substrate. All experiments were performed in triplicate.
Proteins were transferred to 0.22μm NC-membranes. Then membranes were blocked in 3% Bovine Serum Albumin (BSA) diluted in TBST buffer at room temperature, and were incubated overnight at 4℃ with primary antibodies (BACE1 [Abcam, UK, 1:2000], β-actin [Cell Signaling Technology, USA, 1:2000], APP [Thermo Fisher, USA, 1:500]). The membranes were then washed in TBST and incubated with secondary antibody for 1 hour at room temperature. The immunoreactivity of the proteins was detected using Chemiluminescent Substrate. All experiments were performed in triplicate.
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