Cells were lysed in equal proportions of immunoprecipitation (IP) buffer (Beyotime, China) with a complete protease inhibitor cocktail (MedChemExpress, USA) on ice for 30 minutes. After centrifugation, the protein samples were acquired and prepared for immunoprecipitation assays. Protein A/G magnetic beads (MedChemExpress, USA) were initially coupled to anti-NLRP3 antibodies (1:30, Abcam, UK) and rotated for 2 hours at 4 ℃. After washing three times, 800 µg of total protein was added and corotated with antibody magnetic bead complex overnight at 4 ℃. Additionally, the equivalent cognate IgG antibody were used as IP controls. Ultimately, the immunoprecipitated proteins were dissolved in 30 µL 1 x loading buffer (Solarbio, China) by boiling at 10 min for Western blot assay.
Complete protease inhibitor cocktail
Manufactured by MedChemExpress
Sourced in China, United States
The Complete Protease Inhibitor Cocktail is a ready-to-use solution designed to inhibit a broad spectrum of serine, cysteine, and metalloproteases. It is suitable for use in various applications, such as cell lysis, protein extraction, and sample preparation, where the preservation of protein integrity is crucial.
Automatically generated - may contain errors
Lab products found in correlation
Protein a g magnetic beads, by MedChemExpress (1 mentions)
Anti nlrp3 antibody, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by MedChemExpress (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Cell Signaling Technology (1 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)
3 protocols using complete protease inhibitor cocktail
1
NLRP3 Immunoprecipitation Protocol
2
Protein Extraction from Malaria Parasites
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Protein extraction from asexual blood parasites, gametocytes, and ookinetes was performed using buffer A (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1% NP‐40, 0.5% sodium deoxycholate, 0.1% SDS) plus 1× complete protease inhibitor cocktail (MedChemExpress, #HY‐K0010) and 1 mM PMSF (MedChemExpress, #HY‐B0496). After ultrasonication, the protein solution was incubated on ice for 30 min before centrifugation at 12,000 g for 10 min at 4°C. The supernatant was applied to the Western blot analysis. Separate gels with different concentrations were prepared according to the molecular weight of the target proteins. Proteins were separated in SDS–PAGE and transferred to PVDF membrane (Millipore, #IPVH00010). The membrane was blocked in 5% skim milk TBST buffer for 60 min at room temperature and incubated with primary antibodies at 4°C overnight. After incubation, the membrane was washed three times with TBST and incubated with horseradish peroxidase‐conjugated goat anti‐rabbit or anti‐mouse antibodies for 60 min at room temperature. The membrane was washed four times in TBST before enhanced chemiluminescence (Pierce, #32109) detection.
3
Retinal Protein Extraction and Western Blot
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Harvested retinas were homogenized for 1 min on ice with a modified RIPA lysis buffer (#9806, Cell Signaling Technology, MA, USA), containing complete protease inhibitor cocktail (#HY-K0010, MedChemExpress, NJ, USA). The lysates were then centrifuged at 15,000 g for 15 min and protein amounts in the supernatants were measured by Bradford assay. Proteins (10–20 μg) were run on a NuPAGE Bis-Tris gel (Invitrogen, CA, USA) and transferred to polyvinylidene difluoride membranes (GE Healthcare Bio-Science, NJ, USA). The membranes were blocked with 5% non-fat dry milk and PBS/0.1% Tween-20 (PBS-T) for 1 h at room temperature and incubated with primary antibodies (sTable 1 ) for overnight at 4 °C. Membrane were washed three times with PBS-T then incubated with horseradish peroxidase-conjugated secondary antibodies (Bio-Rad, CA, USA) for 1 h at room temperature. Membranes were developed using enhanced chemiluminescence substrate system. The images were captured using a UVP imaging system (UVP LLC, CA, USA).
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