For proteomic and WB analyses, astrocytes were washed twice with phosphate buffer saline (PBS), lysed in 60 µL of buffer O (glycerol 10% (w/v), sodium dodecyl-sulphate (SDS) 2% (w/v), Tris/HCl 62.5 mM, pH 6.8, and cocktails of protease and phosphatase inhibitors (Roche, Basel, Switzerland)) and centrifuged (10 min, 14,000× g, 4 °C) to remove cell debris. Protein concentration was determined in the supernatants with a Lowry assay kit (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s instructions. For WB analyses, samples were diluted to an equal protein concentration using reducing buffer O (i.e., added with dithiothreitol (DTT) (50 mM, final concentration (f.c.) and blue di-bromophenol (0.1% (w/v), f.c.)), and boiled (5 min).
Cocktails of protease and phosphatase inhibitors
Manufactured by Roche
Sourced in Switzerland
Cocktails of protease and phosphatase inhibitors are a type of lab equipment used to preserve the integrity of proteins and prevent unwanted modifications during sample preparation and analysis. They contain a combination of chemicals that block the activity of enzymes that can degrade or alter the structure of proteins.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using cocktails of protease and phosphatase inhibitors
1
Astrocyte Protein Extraction and Quantification
2
Isolating Hippocampal Membrane Fractions
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Hippocampal slices were rapidly washed in cold PBS and then homogenized in a lysis buffer containing 25 mM MES, 2 mM EDTA, and cocktails of protease and phosphatase inhibitors obtained from Roche. After suspension, samples were spun at 100,000 g for 2 h at 4°C. Pellet (plasma membrane fraction, resuspended in PBS containing 0.1% SDS) and supernatant (microsomal fraction) were analyzed in parallel. Protein amount was quantified by Bradford Bicinchoninic Acid Assay kit obtained from Thermo Fisher Scientific, and the cholesterol content was measured per microgram of protein using the Amplex Red Cholesterol Assay kit obtained from Invitrogen.
3
Protein Expression Analysis by Western Blot
4
PrP Protein Extraction and Analysis
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Cells were washed in PBS/Ca/Mg and incubated for 30 min at 4 °C in lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and cocktails of protease and phosphatase inhibitors [Roche, Basel, Switzerland]). After centrifugation of the lysate (14,000 × g, 30 min), the concentration of the proteins in the supernatant was measured with the bicinchoninic acid method (Pierce, Rockford, IL, USA).
For the PNGase assays, 15 µg protein extracts were incubated with 500 U peptide N-glycosidase F (New England Biolabs, Ipswitch, MA, USA) for 1 h at 37 °C. Ten micrograms of proteins were resolved by SDS/10% PAGE and transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL, USA). Membranes were blocked with 3% non-fat dry milk in PBS 0.1% Tween 20 for 1 h at RT and then incubated overnight at 4 °C with 0.02 µg ml−1 Sha31 primary anti-PrP antibody. Bound antibody was revealed by enhanced chemiluminescence detection using a mouse secondary antibody coupled to HRP (GE Healthcare, UK).
For the PNGase assays, 15 µg protein extracts were incubated with 500 U peptide N-glycosidase F (New England Biolabs, Ipswitch, MA, USA) for 1 h at 37 °C. Ten micrograms of proteins were resolved by SDS/10% PAGE and transferred to nitrocellulose membranes (Amersham, Arlington Heights, IL, USA). Membranes were blocked with 3% non-fat dry milk in PBS 0.1% Tween 20 for 1 h at RT and then incubated overnight at 4 °C with 0.02 µg ml−1 Sha31 primary anti-PrP antibody. Bound antibody was revealed by enhanced chemiluminescence detection using a mouse secondary antibody coupled to HRP (GE Healthcare, UK).
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