Frozen tissue samples were crushed and resuspended in LPL assay buffer (25 mM NH4Cl, 5 mM EDTA, 0.01% SDS, 45 U/mL heparin, 0.05% 3-(N,N-Dimethylmyristylammonio) propanesulfonate zwittergent detergent (Acros Organics, 427740050)) containing Mammalian ProteaseArrest (GBiosciences, cat no. 786-331). The tissue suspension was mixed by vortexing and incubated on ice for 30 min, with intermittent disruption with surgical scissors. The resulting lysate was centrifuged at 15,000×g for 15 min at 4 °C to pellet cellular debris. Lipase activity assays were performed on the supernatants as previously described [23] (link); supernatants were combined with a working buffer composed of 0.6 M NaCl, 80 mM Tris–HCl pH 8, 6% fatty-acid free BSA and an EnzChek lipase fluorescent substrate (Molecular Probes, E33955). Fluorescence was measured over 30 min at 37 °C on a SpectraMax i3 plate reader (Molecular Devices). Relative lipase activity was calculated by subtracting background (calculated by reading fluorescence of a sample with no LPL) and then calculating the slope of the curve between the 5 and 13 min reads. The data were graphed as the average of slopes for each group.
Mammalian proteasearrest
Manufactured by G Biosciences
Mammalian ProteaseArrest is a buffer solution designed to inhibit proteases commonly found in mammalian cell and tissue samples. It contains a mixture of protease inhibitors that target a broad spectrum of protease activities.
Automatically generated - may contain errors
Lab products found in correlation
Enzchek lipase fluorescent substrate, by Thermo Fisher Scientific (1 mentions)
Spectramax i3 plate reader, by Molecular Devices (1 mentions)
Pierce bicinchoninic acid assay, by Thermo Fisher Scientific (1 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg h l, by Abcam (1 mentions)
Cyp2e1, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Cyp3a4, by Abcam (1 mentions)
Cyp1a2, by Abcam (1 mentions)
Micro bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
4 protocols using mammalian proteasearrest
1
Measurement of Lipase Activity in Frozen Tissue
2
Measurement of Brain Cytokines
3
Characterization of CYP Enzymes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After transfections, cells were lysed with ice-cold RIPA lysis buffer (ThermoFisher Scientific, Carlsbad, CA) containing protease inhibitors (Pierce Protease Inhibitor Mini Tablets, Rockford, IL and Mammalian Protease Arrest, GBiosciences, St. Louis, MO) and centrifuged at 12,500 rpm for 10 min at 4 °C. Cell supernatants were used for immunoblots. Proteins were quantified using Pierce BCA Protein Assay Kit (ThermoFisher Scientific, Carlsbad, CA) and 20 ug of protein was loaded on to precast polyacrylamide Bis-tris 10% gels (GenScript, Piscataway, NJ). The membranes were blocked with 5% skim milk and incubated with primary antibodies against CYP2E1, CYP1A2 and CYP3A4 (Abcam, Cambridge, MA) and β-actin (Sigma, St. Louis, MO). Next membranes were incubated with HRP-conjugated secondary antibodies to goat anti-rabbit IgG H&L (Abcam, Cambridge, MA). The protein bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher Scientific, Carlsbad, CA) and densitometry was performed with the ImageJ software program (NIH, Bethesda, MD).
4
Protein Expression Analysis in Cell Lysates
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To analyze the expression of proteins, cells were lysed directly on a plate in 100 μl cold RIPA–EDTA buffer [50 mmol/L Tris–HCl, 150 mmol/L NaCl, 0.5% NP-40, 0.5% sodium deoxycholate, 0.5% Triton™ X-100, 0.1% SDS, and 5 mmol/L EDTA, pH 7.4] containing Phosphatase Inhibitor Cocktails 2 and 3 (Sigma, P5726 and P0044), and protease inhibitor (Mammalian ProteaseArrest™, GBiosciences, A Geno Technology Inc.). Cells were incubated on ice for 5 min, scraped, transferred to the Eppendorf, pipetted 10 times with 200 μl tip and centrifuged at 10,000×g at 4 °C for 5 min. The supernatants were collected and then protein concentration was determined using Micro BCA™ Protein Assay Kit (Thermo Scientific, Pierce).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!