Western blotting was performed, as described previously.[18 (link)] BMMs were incubated in the same manner as real-time PCR assays. The cells were washed with phosphate-buffered saline and lysed in a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, and 1% deoxycholate) supplemented with 1 mM phenylmethylsulfonyl fluoride (Bio Basic Inc., Amherst, NY, USA). The lysates were centrifuged at 20,000×g for 13 min at 4℃ and the supernatant containing the proteins was collected. The proteins were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Billerica, MA, USA). The membranes were blocked with 5% skim milk for 1 hr at room temperature and incubated overnight at 4℃ with the primary antibody. They were then incubated with the secondary antibody conjugated to horseradish peroxidase for 2 hr at room temperature. The membranes were developed with CLAROTM Mucho (Bio-D, Gwangmyeong, Korea) using a LAS-4000 luminescent image analyzer (Fuji Photo Film Co. Ltd., Tokyo, Japan).
Phenylmethylsulfonyl fluoride
Manufactured by Bio Basic
Sourced in United States
Phenylmethylsulfonyl fluoride (PMSF) is a protease inhibitor commonly used in laboratory settings. It is a colorless, crystalline solid that is soluble in organic solvents. PMSF functions by irreversibly inhibiting a wide range of serine proteases, including trypsin, chymotrypsin, and thrombin, making it useful in preventing protein degradation during sample preparation and analysis.
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Lab products found in correlation
2 protocols using phenylmethylsulfonyl fluoride
1
Western Blotting Protocol for Protein Analysis
2
Expression and Purification of Mx1-9R Protein
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Transformed BL21 cells were cultured in Luria-Bertani (LB) broth (MB Cell, Los Angeles, CA, USA) containing 30 μg/mL of kanamycin (LPS solution, Daejeon, Korea) for 3 h at 37 °C, then incubated with 0.1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Goldbio, St. Louis, MO, USA) at 18 °C overnight. After IPTG induction, cells were centrifuged and resuspended in lysis buffer containing 50 mM of Tris-HCl (Welgene, Daegu, Korea), 0.5 M of NaCl (Welgene), and 10 mM of imidazole (Biosesang, Seongnam, Korea) with 1 mM of phenylmethylsulfonyl fluoride (Biobasic, Toronto, ON, Canada). Cell suspension was lysed by sonication for 40 × 10 s with 10 s intervals on ice. The lysate was centrifuged at 8000 rpm for 30 min at 4 °C, and His-tagged Mx1-9R was purified by Ni-NTA resin (Incospharm, Daejeon, Korea). Then, purified Mx1-9R fusion proteins were desalted using Amicon Ultra-15 Centrifugal Filter Units (Millipore Sigma, Darmstadt, Germany). Supernatant, pellet, flow through, and eluent were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gel was stained with 0.05% Coomassie Blue R-250 (LPS solution). The amounts of recombinant proteins were quantified by Bradford assay or NanoDrop (Thermo Fisher, Waltham, MA, USA) measurements.
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