Cells were collected and lysed in radioimmunoprecipitation assay buffer (Cell Signaling Technology) containing 1% Protease Inhibitor Cocktail (MCE) and 1% Phosphatase Inhibitor Cocktail (MCE) or EpiQuik Total Histone Extraction Kit (EpiGentek), and protein concentration was determined using the BCA Protein Assay Kit (Thermo Fisher Scientific). Protein extracts were separated on 8 to 15% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) by electrophoresis and transfer equipment (Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore). The PVDF membrane was blocked and incubated with the corresponding primary and secondary antibodies, reacted with enhanced chemiluminescence (ECL) detection reagent (PerkinElmer), and exposed to x-ray films or imaging system (Azure). For the detection of proteins with the same or similar molecular weight, the primary antibody was stripped with ReBlot Plus Strong Antibody Stripping Solution (Millipore), and the corresponding primary and secondary antibodies were reincubated for chemiluminescence. Results were normalized to the internal control β-actin or histone 3. All antibody information is shown in table S1.
Protease inhibitor cocktail
Manufactured by Epigentek
Sourced in United States
Protease Inhibitor Cocktail is a ready-to-use solution designed to inhibit a broad spectrum of serine, cysteine, and metalloproteinases. It is intended for use in protein extraction and purification procedures to prevent degradation of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
3 mbz, by Merck Group (3 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (3 mentions)
Bioruptor 300, by Diagenode (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Bza benzylamine, by Merck Group (2 mentions)
Radioimmunoprecipitation assay buffer, by Cell Signaling Technology (1 mentions)
Reblot plus strong antibody stripping solution, by Merck Group (1 mentions)
Ecl detection reagent, by PerkinElmer (1 mentions)
Epiquik total histone extraction kit, by Epigentek (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
4 protocols using protease inhibitor cocktail
1
Protein Extraction and Western Blot Analysis
2
Drosophila S2 Cell Lysis and Co-IP
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A total of 107 Drosophila melanogaster S2 cells were pelleted and resuspended in 0.5 mL of RIPA buffer, to which protease inhibitors were added: 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma, St Louis, MO, USA, #10837091001), 1× protease inhibitor cocktail (EpiGentek, Farmingdale, NY, USA, #R-1101), 10 mM 3-MBZ (3-Methoxybenzylamine, Sigma, #159891), and 0.5 mM BZA (Benzylamine, Sigma, #185701). Cell suspension was put on ice for 30 min, followed by 6 cycles of sonication (each cycle: 10 s on/10 s off) on a Bioruptor300 (Diagenode, Denville, NJ, USA). The cell lysate was cleared by spinning at 13,000 rpm for 10 min. The resultant supernatant was then used for co-immunoprecipitation and Western blot analyses.
3
Protein and RNA Extraction from S2 Cells
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The S2 cells were pelleted, washed, and resuspended in 0.5 mL RIPA buffer supplemented with 1 mM PMSF (phenylmethylsulfonyl fluoride, Sigma-Aldrich, St. Louis, MO, USA, 68178, #10837091001), 1X protease inhibitor cocktail (EpiGentek, Farmingdale, NY, #R-1101), 10 mM 3-MBZ (3-methoxybenzylamine, Sigma-Aldrich, St. Louis, MO, USA, 68178, #159891), and 0.5 mM BZA (benzylamine, Sigma-Aldrich, St. Louis, MO, USA, 68178, #185701). The cell suspension was incubated on ice for 30 min, gently vortexed, sonicated for 6 cycles (10 s on/10 s off) (Bioruptor300, Diagenode, Denville, NJ, USA), and spun down at 13,000 rpm for 10 min. The supernatant was used for Western blot analyses. The total RNA was extracted from the washed and pelleted cells using the Quick-RNA Miniprep Kit (Cat. #R1054) as per the manufacturer’s protocol (Zymo Research, Irvine, CA, USA, 92614) and eluted in RNase-free water. The total RNA was submitted (Novogene, Sacramento, CA, USA, 95826) for ribosomal-depleted paired-end RNA sequencing.
4
Drosophila S2 Cell Lysis and Fractionation
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Drosophila melanogaster S2 cells 107 were resuspended in 0.5 ml of RIPA buffer with fresh added inhibitors: 1 mM PMSF (Phenylmethylsulfonyl fluoride, Sigma, St Louis, MO, #10837091001), 1xProtease inhibitor cocktail (EpiGentek, Farmingdale, NY, #R-1101), 10 mM 3-MBZ (3-Methoxybenzylamine, Sigma, #159891) and 0.5 mM BZA (Benzylamine, Sigma, #185701), incubated for 30 min on ice with mild vortexing, and then sonicated for 6 cycles (10 sec on/10 sec off (Bioruptor300, Diagenode, Denville, MJ) and spun down at 13000 rpm for 10 min. Supernatant was used for Western blot analyses.
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