Pefablock

Manufactured by Merck Group

Sourced in France

Pefablock is a laboratory equipment product manufactured by Merck Group. It is designed to perform specific functions in a laboratory setting. The core function of Pefablock is to provide a reliable and efficient means of handling and processing samples.

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Pefablock SC

6 protocols using pefablock

Antibody-based Protein Detection Protocol

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The following antibodies were obtained from commercial sources: monoclonal mouse anti-CD59 (MEM-43, Abcam, Cat. No. ab9182), monoclonal rabbit anti-flotillin-1 (D2V7J) XP (Cell Signaling, Cat. No. 18634), monoclonal mouse anti-flotillin-2 (BD Biosciences, Cat. No. 610383), polyclonal rabbit anti-GAPDH (Sigma, Cat. No. G9545), monoclonal rabbit anti-phospho-Src Family (Tyr⁴¹⁶, Cell Signaling, Cat. No. 6943), monoclonal rabbit anti-Src Family (Cell Signaling, Cat. No. 2109), monoclonal rabbit anti-caveolin-1 (D46G3) XP (Cell Signaling, Cat. No. 3267). LecA was detected by a custom-made polyclonal rabbit anti-LecA antibody (Eurogentec, France).
The protease inhibitors Aprotinin, Leupeptin, Pefablock, Sodium orthovanadate and Phosphatase Inhibitor Cocktail 3 were all obtained from Sigma-Aldrich. RPMI 1640, DPBS-/-, FCS and l-Glutamine were all purchased from Gibco (Thermo Fisher Scientific). The following chemicals were obtained from Roth: BSA, DABCO, DAPI, EDTA, glycerol, LB, Mowiol, NaCl, sodium deoxycholate, NH₄Cl, paraformaldehyde, SDS, Tris (hydroxymethyl)-aminoethane, Triton X-100 and Tween 20. Amikacin sulphate and Gentamicin were obtained from Sigma-Aldrich. StxB was purchased from Sigma-Aldrich (Cat. No. SML0562).

Brandel A., Aigal S., Lagies S., Schlimpert M., Meléndez A.V., Xu M., Lehmann A., Hummel D., Fisch D., Madl J., Eierhoff T., Kammerer B, & Römer W. (2021). The Gb3-enriched CD59/flotillin plasma membrane domain regulates host cell invasion by Pseudomonas aeruginosa. Cellular and Molecular Life Sciences, 78(7), 3637-3656.

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Unconjugated Bilirubin Binding Assay

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Unconjugated bilirubin and bilirubin oxidase were purchased from Sigma-Aldrich (St. Louis, MO). Fluorecene-5’-maleimide and DyLight633-maleimide were from Thermo Scientific (Waltham, MA). Metal ion chelating resin was purchased from GE Healthcare Life sciences (Mickleton, NJ). Pooled normal human plasma (NHP) was obtained from George King Biotechnology (Overland Park, KS). SeaKem HGT (P) agarose (Lonza, Rockland, ME), nitrocellulose membrane (Bio-Rad, Hercules, CA), Pefablock (Sigma-Aldrich), rabbit anti-human VWF antibody (Dako, Carpinteria, CA), and IRDye-800CW-labeled goat anti-rabbit IgG (LI-COR Biotech, Lincoln, NE) were all commercially available. Plasma VWF [15 (link)] and recombinant human ADAMTS13 (rA13) [16 (link)] were purified to homogeneity using the methods previously described. GST-VWF73-H [17 (link)] and GST-rVWF71-11K [8 (link)] were prepared according to methods described previously. Leftover de-identified plasma samples anti-coagulated with sodium heparin after chemistry tests were collected from hospitalized patients with significantly elevated serum levels of Unconjugated bilirubin.

Lu R.N., Yang S., Wu H.M, & Zheng X.L. (2015). Unconjugated Bilirubin Inhibits Proteolytic Cleavage of von Willebrand Factor by ADAMTS13 Protease. Journal of thrombosis and haemostasis : JTH, 13(6), 1064-1072.

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Chromatin Proteolysis Assay Protocol

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Chromatin was extracted using the Subcellular Protein Fractionation Kit for Culture Cells (Thermo Fisher Scientific, 78840) according to the manufactures instructions with the exception that the provided protease inhibitors were substituted with 0.5 mM pefablock (Sigma-Aldrich, 11429868001). Equal amounts of chromatin (30 µg protein) were incubated at 37 °C for 15 min in a pH-modified cathepsin reaction buffer (50 mM sodium acetate, 4 mM EDTA, pH 7.0), containing 0.5 mM pefabloc, 5 mM dithiothreitol (Sigma-Aldrich, D9779), and indicated amounts of rCTSB (Biovison, 7580), rCTSL (Biovison, 1135) and ALLN (Calbiochem, 208719). Samples were subsequently boiled for 5 min and analyzed by immunoblotting.

Hämälistö S., Stahl J.L., Favaro E., Yang Q., Liu B., Christoffersen L., Loos B., Guasch Boldú C., Joyce J.A., Reinheckel T., Barisic M, & Jäättelä M. (2020). Spatially and temporally defined lysosomal leakage facilitates mitotic chromosome segregation. Nature Communications, 11, 229.

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Biotinylation and Detection of AQP4 Protein

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Oocytes used for water permeability measurements were subsequently exposed to sulfo-NHS-SS-biotin for 45 min at 4°C and solubilized in lysis buffer (150 mM NaCl, 50 mM Tris/HCl, 1% (w/v) Triton X-100, 0.05% SDS, 5 mM EDTA, and protease inhibitors [0.4 mM pefablock, 8 µM leupeptin; Sigma-Aldrich]) for 30 min on ice. The resulting lysates were sonicated and centrifuged at 10,000g for 5 min at 4°C after which the supernatant was incubated with Neutravidin beads (VWR) for 60 min at room temperature (RT). After a brief centrifugation (1000g for 1 min), the supernatant was removed and the beads washed to remove unspecific proteins from the bead slurry. The biotinylated proteins were retrieved by incubation with SDS sample buffer (4% SDS, 0.006% bromophenol blue, 8.7 % glycerol, 0.25% Tris-base and 0.75% DTT [pH 6.8, adjusted with HCl]), electrophoresed on SDS/PAGE (12%), and transferred to PVDF membranes. Proteins were detected by use of a primary polyclonal anti-AQP4 antibody from Alomone labs (#AQP4-004) and a secondary antibody from Licor Biosciences (Licor 800; 1:10.000). The membranes were scanned (Licor) to allow for visualization of immunoreactive bands. Protein abundances were analyzed by densitometry using Image Studio 5.2 software (Licor).

Berland S., Toft-Bertelsen T.L., Aukrust I., Byska J., Vaudel M., Bindoff L.A., MacAulay N, & Houge G. (2018). A de novo Ser111Thr variant in aquaporin-4 in a patient with intellectual disability, transient signs of brain ischemia, transient cardiac hypertrophy, and progressive gait disturbance. Cold Spring Harbor Molecular Case Studies, 4(1), a002303.

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Efficient Nuclear Fractionation Protocol

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Cells were seeded, starved, and stimulated as described above, washed two times with cold PBS, lysed in 1 ml of cytoplasmic lysis buffer (10 mM MES, pH 6.2, 10 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, 5 mM DTT, 1% Triton X-100, 1 mM pefablock [Sigma], and 1 mM NaF) for 10 min on ice, scraped into Eppendorf tubes, and centrifuged for 10 min at 3,000 rpm in a bench-top centrifuge. The supernatant was recentrifuged at 13,000 rpm for 20 min and collected as the cytoplasmic fraction. The pellet from the first centrifugation (nuclei) was washed three times with 1 ml of cytoplasmic lysis buffer supplemented with 1% NP-40 and once with cytoplasmic lysis buffer without addition of detergents. The purified nuclear pellet was resuspended in 0.5 ml of nuclear extraction buffer (25 mM Tris-HCl, pH 10.5, 1 mM EDTA, 0.5 M NaCl, 5 mM β-mercaptoethanol, and 0.5% Triton X-100), vortexed for 10 min at 4°C, and centrifuged for 20 min at 13,000 rpm, and the supernatant was collected as a nuclear fraction. This protocol was specifically optimized and regularly tested for the nuclear fraction not to contain any contamination of Golgi, ER, lipid rafts, and other organelles.

Papadopoulos N., Lennartsson J, & Heldin C.H. (2018). PDGFRβ translocates to the nucleus and regulates chromatin remodeling via TATA element–modifying factor 1. The Journal of Cell Biology, 217(5), 1701-1717.

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Mouse Neuronal Cytoplasmic Protein Analysis

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Dissected mouse CP were dissolved in ice-cold dissection buffer containing 0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, 8.4 μM leupeptin (Calbiochem), and 0.4 mM Pefabloc (Roche), with pH 7.2, and sonicated using a probe sonicator (BioLogics Inc. 150 V/T, 3 × 5 bursts at 60% power). For SDS-PAGE, protein contents were quantified (Pierce BCA Protein Assay Kit) and samples were adjusted to 1.5% (w/v) sodium dodecyl sulfate, 40.0 mM 1,4-dithiothreitol, 6% (v/v) glycerol, and 10 mM Tris, pH 6.8 with bromophenol blue. The samples were heated at 65 °C for 15 min and approximately 10 µg of protein per sample was separated by 12.5% polyacrylamide gel electrophoresis.
For native PAGE, homogenates were added protease inhibitors (Pefablock, leupeptin, Sigma-Aldrich) and non-denaturing gel sample buffer, 1% digitonin, and sample additive (NativePAGE Sample Prep Kit, Invitrogen). After centrifugation at 20,000× g for 30 min at 4 °C, soluble protein samples were loaded onto 3–12% Bi-Tris gels (NativePAGE, Invitrogen) and electrophoresed at 150 V for 1 h at 4 °C according to the manufacturers protocol.

Baasch Christensen I., Cheng L., Brewer J.R., Bartsch U., Fenton R.A., Damkier H.H, & Praetorius J. (2021). Multiple Na,K-ATPase Subunits Colocalize in the Brush Border of Mouse Choroid Plexus Epithelial Cells. International Journal of Molecular Sciences, 22(4), 1569.

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