Stationary-phase cultures of wild-type P. gingivalis and mutants were adjusted to an optical density at 600 nm (OD600) of 1.0, and cells were collected by centrifugation (8,000 × g, 15 min). The cell pellet was then washed and resuspended in phosphate-buffered saline (PBS). This fraction is referred to as washed cells (WC). The collected cell-free culture medium was ultracentrifuged (100,000 × g, 1 h) to remove vesicles, and the supernatant was concentrated 10-fold by ultrafiltration using 3-kDa cutoff centricones (EMD, Millipore, Billerica, MA); this fraction was designated as the clarified medium (CF). The WC fraction, obtained as described above, was suspended in buffer containing 0.25 M sucrose and 30 mM Tris-HCl at pH 7.6. After a 10-min incubation period, the cells were pelleted (12,500 × g, 15 min) and rapidly resuspended in 2.5 ml of cold distilled water to disrupt the OM. After an additional 10-min incubation period, spheroplasts were pelleted by centrifugation (12,500 × g, 15 min). The supernatant was collected and designated as the periplasmic (PP) fraction. All fractions were supplemented with peptidase inhibitors (5 mM tosyl-l -lysyl-chloromethyl ketone [TLCK], 1 mM 2,2′-dithiodipyridine [DTDP], 1× EDTA-free protein inhibitor cocktail; all from Roche) before storage at –20°C.
Edta free protein inhibitor cocktail
Manufactured by Roche
EDTA-free protein inhibitor cocktail is a laboratory reagent designed to inhibit protease activity in protein samples. It provides a broad-spectrum inhibition of serine, cysteine, and metalloproteases without the use of EDTA. The product is intended for use in research and analytical applications where protease inhibition is required.
Automatically generated - may contain errors
5 protocols using edta free protein inhibitor cocktail
1
Fractionation of P. gingivalis Cells
2
Acetylation of α-synuclein and S100A11
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α-synuclein and S100A11 recombinant proteins were incubated in the presence of recombinant KAT2A, KAT2A mut, KAT2B or KAT2B mut, separately. The reaction mixture (25 μl) containing 1× HAT buffer (50 mM Tris-HCl pH 7.9, 7% glycerol, 0.1 mM EDTA, 50 mM KCl, 1 mM DTT), 100 mM sodium butyrate, 0.3 mM Acetyl-CoA, 1× EDTA free protein inhibitor cocktail (Roche) at final concentration was incubated for 1h at 30°C. The reaction was stopped by adding Laemmli buffer with 10 mM DTT and boiled for 5–10 min. Proteins from the reactions were separated on a 4-12% SDS–PAGE and tested by western blot analyses.
3
Acetylation of α-synuclein and S100A11
4
Reagents and Antibodies for Cell Culture Experiments
5
Oxidative Stress Response in Alkbh8 Knockout
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Alkbh8-/- and wt MEF cells were cultured in 6-well format and treated with 1.2 mM H2O2 in serum-free media for 1 h. After treatment media was replaced with complete growth media and incubated for 48 h in a humidified CO2-incubator. Cells were harvested and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, with 150 mM sodium chloride, 1% TritonX-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) supplemented with an EDTA-free protein inhibitor cocktail (Roche Diagnostics). Extracted protein was normalized using Bradford (BioRad) analysis according to manufacturer’s instruction. LPO was analyzed using the 8-Isoprostane EIA assay kit from Cayman Chemical Company (Ann Arbor, MI, USA). The 96-well assay plate was set up and run as suggested by the manufacturer, using an eight point standard curve run in duplicate. Protein samples were diluted in EIA buffer (supplied by the manufacturer) to a final concentration of 0.250 μg per well. Data reported comes from biological triplicates, with each sample assayed in duplicate. The 8-isoprostane sample concentrations from the mean absorbance were interpolated from a sigmoid standard curve (4 parameter logistic nonlinear regression) using Graph Pad software. A Student t-test was used to identify significant differences between groups at p < 0.05.
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Alkbh8-/- and wt MEF cells were cultured in 6-well format and treated with 1.2 mM H2O2 in serum-free media for 1 h. After treatment media was replaced with complete growth media and incubated for 48 h in a humidified CO2-incubator. Cells were harvested and lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4, with 150 mM sodium chloride, 1% TritonX-100, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate) supplemented with an EDTA-free protein inhibitor cocktail (Roche Diagnostics). Extracted protein was normalized using Bradford (BioRad) analysis according to manufacturer’s instruction. LPO was analyzed using the 8-Isoprostane EIA assay kit from Cayman Chemical Company (Ann Arbor, MI, USA). The 96-well assay plate was set up and run as suggested by the manufacturer, using an eight point standard curve run in duplicate. Protein samples were diluted in EIA buffer (supplied by the manufacturer) to a final concentration of 0.250 μg per well. Data reported comes from biological triplicates, with each sample assayed in duplicate. The 8-isoprostane sample concentrations from the mean absorbance were interpolated from a sigmoid standard curve (4 parameter logistic nonlinear regression) using Graph Pad software. A Student t-test was used to identify significant differences between groups at p < 0.05.
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