To extract total protein, transfected cells were lysed with radioimmunoprecipitation assay (RIPA) solution (Sangon Biotech Co. Ltd., Shanghai, China) supplemented with a protease inhibitor cocktail (Sangon Biotech Co. Ltd.) and phenylmethanesulfonyl fluoride (Sangon Biotech Co. Ltd.). After total protein quantification using a BCA Protein Assay Kit (Sangon Biotech Co. Ltd.), equal amounts of protein were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk at room temperature for 2 h and subsequently incubated overnight at 4°C with primary antibodies against SP1 (Cat. No. b124804; Abcam, Cambridge, MA, USA) or GAPDH (Cat. No. ab128915; Abcam). The primary antibodies were used with a dilution of 1:1,000. Next, a goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Cat. No. ab6721; Abcam) was incubated with the membranes at room temperature for 2 h. Finally, the protein signals were visualized using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, IL, USA).
Phenylmethanesulfonyl fluoride
Manufactured by Sangon
Sourced in China
Phenylmethanesulfonyl fluoride is a chemical compound commonly used as a protease inhibitor in biochemical research. It functions by irreversibly inhibiting serine proteases, which are enzymes that cleave peptide bonds in proteins. This property makes Phenylmethanesulfonyl fluoride a valuable tool for researchers studying protein structure and function.
Automatically generated - may contain errors
Lab products found in correlation
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Ripa solution, by Sangon (1 mentions)
Gapdh, by Abcam (1 mentions)
Bca protein assay kit, by Sangon (1 mentions)
Protease inhibitor cocktail, by Sangon (1 mentions)
Bicinchoninic acid protein assay kit, by Sangon (1 mentions)
Enhanced chemiluminescence detection reagent, by Merck Group (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Gel imaging analysis system, by Bio-Rad (1 mentions)
Protease inhibitors cocktail, by Sangon (1 mentions)
Bradford reagent, by Merck Group (1 mentions)
Protein a g agarose, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
4 protocols using phenylmethanesulfonyl fluoride
1
Protein Extraction and Western Blot Analysis
2
Western Blot Analysis of MMP1 Protein
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Cultured cells were washed 3 times with PBS and lysed in phenylmethanesulfonyl fluoride (Sangon Biotech Co., Ltd., Shanghai, China) on ice for 20 min. The lysates were centrifuged at 15,000 × g at 4°C for 10 min and the supernatants were collected. The protein concentration was measured using a Bicinchoninic Acid protein assay kit (Sangon Biotech Co., Ltd.), with 2 mg/ml BSA (Gibco; Thermo Fisher Scientific, Inc.) as the standard. Subsequently, 50 µg proteins were separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Merck KGaA). The membranes were blocked in 5% nonfat dried milk at room temperature for 1 h, and then incubated at 4°C with primary antibodies (anti-MMP1 antibody; 1:10,000; cat. no. sc-58377; Santa Cruz Biotechnology, Inc., Dallas, TX, USA and anti-β-actin antibody, 1:10,000; cat. no. 115035003; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) overnight. Following washing with Tris buffered saline with Tween-20 (1:1,000) 4 times, the blots were incubated with horseradish peroxidase-conjugated goat-anti-rabbit antibody (1:5,000; cat. no. 111035047; Jackson ImmunoResearch Laboratories, Inc.) at 37°C for 1 h, and then developed with enhanced chemiluminescence detection reagents (Merck KGaA). Visualization was performed with a gel imaging analysis system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
3
Western Blot Analysis of NUAK1 Protein
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Mouse cortical tissues or cultured cells were lysed in RIPA buffer [150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5% deoxycholic acid, 0.1% (w/v) sodium dodecyl sulfate (SDS)] containing protease inhibitors (cocktail, Sangon) and 1 mM phenylmethanesulfonyl fluoride (Sigma) for 15 min. The lysates were centrifuged at 14000 × g for 10 min at 4°C. Protein samples (supernatants) were immediately transferred into new tubes, and the concentration was measured using Bradford Reagent (Sigma). After boiling for 10 min at 98°C, the samples were separated on SDS–polyacrylamide gel electrophoresis (PAGE) gels followed by transferring onto nitrocellulose membranes (PALL). The membranes were treated with the primary antibodies against NUAK1 (Cell Signaling Technology, rabbit polyclonal antibody) and GAPDH (Sangon, rabbit polyclonal antibody) overnight at 4°C following blocking with 5% (w/v) skim milk in TBS-T (Tris-buffered saline containing 0.1% Tween-20) for 1 h. Next, the membrane was washed with TBS-T buffer three times and incubated with infrared-labeled secondary antibodies (Odyssey, Goat anti-rabbit IR Dye @ 680 CW) at room temperature for 2 h. After washing, the membranes were finally scanned with Odyssey v3.0. The intensity of each band was quantified using Image J analysis.
4
Coimmunoprecipitation of PRMT5 interactors
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Coimmunoprecipitation was carried out according to a previously reported method in the laboratory61 (link). Briefly, G. lucidum mycelia powder (0.15 g) was lysed in lysis buffer containing 100 mM NaCl, 20 mM Tris–HCl pH 7.6, 0.1% Triton X-100, 0.5% protease inhibitor cocktail (Sigma), and 1 mM phenylmethanesulfonyl fluoride (Sangon) for 1 h. The supernatants (200 µL) were immunoprecipitated with protein A/G agarose (25 µL, Thermo Scientific, 88802), followed by incubation with control rabbit IgG (Solarbio) or PRMT5 antibody (Abcam, ab109451) at 4 °C overnight. The immunoprecipitated proteins were was washed (5 times) with lysis buffer, and the protein lysate was further eluted with 5× SDS loading buffer. Western blotting was further performed using anti-PRMT5 antibody and anti-GlPP2C1 polyclonal antibody. In addition, using the same method to immunoprecipitate interacting proteins with PRMT5, the proteins were separated on a 12% (wt/vol) SDS‒PAGE gel and stained with Coomassie blue. Bands were excised, followed by digestion and plastid analysis.
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