Pefabloc proteinase inhibitor

Manufactured by Avantor

Sourced in United States

Pefabloc is a proteinase inhibitor that inactivates a broad range of serine proteases. It is commonly used in research applications to prevent protein degradation during sample preparation and analysis.

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Lab products found in correlation

Horseradish peroxidase conjugated goat anti mouse igg antibody, by Merck Group (2 mentions) Pvdf membrane, by Merck Group (2 mentions) Super rx, by Fujifilm (2 mentions) Fluorichemq, by Bio-Techne (1 mentions) Fluochemq software, by Bio-Techne (1 mentions) Lysotracker red dnd 99, by Thermo Fisher Scientific (1 mentions) Proteinase k, by Avantor (1 mentions) Proteinase k, by Roche (1 mentions) Nupage bis tris gel, by Thermo Fisher Scientific (1 mentions)

4 protocols using pefabloc proteinase inhibitor

PrP Detection in Brain Extracts

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For PrP analysis in brain extracts, brain homogenates (3 different animals) prepared in PBS were either not digested or treated with different concentrations of PK (0 to 5 mg/ml; VWR, Ca) as indicated for one hour at 37°C. The reaction was terminated by adding 1X pefabloc proteinase inhibitor (VWR, Ca). Fifty μg of protein were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and then electrophoretically transferred to PVDF membranes (Millipore, Ca). PVDF membranes were probed using anti-PrP monoclonal antibodies followed by horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Sigma, Ca) and developed using ECL-plus detection (Amersham). Images were acquired on X-ray film (Super Rx; Fujifilm) or by using a digital imaging system (Alpha Innotech, FluoriChemQ). FluoChemQ software (Alpha Innotech) was used to quantify and determine the relative values of PrP^res signals.

Hannaoui S., Amidian S., Cheng Y.C., Duque Velásquez C., Dorosh L., Law S., Telling G., Stepanova M., McKenzie D., Wille H, & Gilch S. (2017). Destabilizing polymorphism in cervid prion protein hydrophobic core determines prion conformation and conversion efficiency. PLoS Pathogens, 13(8), e1006553.

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Detecting Misfolded Proteins in Brain Homogenates

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Brain homogenates (10% w/v) prepared in PBS were digested or treated with different concentrations of PK (0–0.5 mg/ml; VWR, Visalia, CA, USA) for 1 h at 37°C. 1× pefabloc proteinase inhibitor (VWR, Visalia, CA, USA) was added to terminate the reaction. The relative amount of misfolded protein was measured by IDEXX HerdChek BSE Kit (see above). Each sample was performed in triplicate.

Hwang S., West Greenlee M.H., Balkema-Buschmann A., Groschup M.H., Nicholson E.M, & Greenlee J.J. (2018). Real-Time Quaking-Induced Conversion Detection of Bovine Spongiform Encephalopathy Prions in a Subclinical Steer. Frontiers in Veterinary Science, 4, 242.

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Immunoblotting of Protein Markers

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Proteinase K and Pefabloc proteinase inhibitor were obtained from VWR. Immunoblotting was done using the enhanced chemiluminescence blotting technique (ECL plus) from Pierce. The monoclonal anti-PrP antibody (mAb) 4H11 has been described30 (link). Polyclonal anti-rab7, rab9 and rab11 antibodies were purchased from Santa Cruz, Alexa488-labelled EGF and lysotracker red dnd-99 were obtained from Molecular Probes. Polyclonal antibodies against EGFR and TRAPα, respectively, were from Abcam. All other chemicals were from Sigma.

Shim S.Y., Karri S., Law S., Schatzl H.M, & Gilch S. (2016). Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells. Scientific Reports, 6, 21658.

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PrP Detection in Brain Extracts

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For PrP analysis in brain extracts of different animal models, brain homogenates prepared in PBS were digested with different concentrations of proteinase K (PK; Roche) for 1 hour at 37°C. The enzymatic reaction was terminated by the addition of 1X pefabloc proteinase inhibitor (VWR), then samples were denatured at 96°C for 10 min in 3X SDS (sodium dodecyl sulphate) sample buffer. For deglycosylation of PK-digested and denatured samples, PNGase F enzyme (Roche) was added to the samples according to the manufacturer’s instructions. Samples were resolved on 12% NuPAGE bis-tris gels (Life Technologies), and then electrophoretically transferred to PVDF membranes (Millipore, Ca). PVDF membranes were probed using anti-PrP monoclonal antibodies followed by horseradish peroxidase-conjugated goat anti-mouse IgG antibody (Sigma) and developed using ECL-plus detection (Amersham). Images were acquired on X-ray film (Super Rx; Fujifilm).

Hannaoui S., Triscott E., Duque Velásquez C., Chang S.C., Arifin M.I., Zemlyankina I., Tang X., Bollinger T., Wille H., McKenzie D, & Gilch S. (2021). New and distinct chronic wasting disease strains associated with cervid polymorphism at codon 116 of the Prnp gene. PLoS Pathogens, 17(7), e1009795.

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