Protease inhibitor

Manufactured by Qiagen

Sourced in Germany

Protease inhibitors are chemical compounds that are used to prevent the activity of proteases, which are enzymes that break down proteins. They are commonly used in various laboratory applications, such as protein purification and cell culture, to preserve the integrity of proteins and prevent their degradation.

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Lab products found in correlation

Qproteome mitochondria isolation kit, by Qiagen (2 mentions) Lysis buffer, by Qiagen (1 mentions) Mitochondria storage buffer, by Qiagen (1 mentions) Ripa lysis buffer, by Beyotime (1 mentions) Protein a magnetic bead, by Merck Group (1 mentions) Tissuelyser 2, by Qiagen (1 mentions) C18 macro spincolumns, by Harvard Apparatus (1 mentions) Image lab software, by Bio-Rad (1 mentions) Ripa buffer, by Beyotime (1 mentions) Ab2302, by Abcam (1 mentions) Ab32124, by Abcam (1 mentions) Ab176323, by Abcam (1 mentions) Ab174830, by Abcam (1 mentions) Ab65596, by Abcam (1 mentions) Ab121000, by Abcam (1 mentions) Bicinchoninic acid (bca), by Thermo Fisher Scientific (1 mentions) Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions) Protease inhibitor, by Roche (1 mentions) Gel filtration standard, by Bio-Rad (1 mentions) S200 increase 10 300 column, by GE Healthcare (1 mentions)

Close spelling variants of this product

Protease inhibitor cocktail (16 citations) COmplete protease inhibitors (4 citations) Protease and phosphatase inhibitors (2 citations)

8 protocols using protease inhibitor

Mitochondrial Protein Extraction from Brain

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Mitochondrial and cytoplasmic proteins were extracted from brain tissue or neuron cell culture using a Qproteome Mitochondria Isolation Kit (Qiagen, Germany).
Cells or freshly excised tissues were homogenized with a lysis buffer (Qiagen, Germany) supplemented with a Protease Inhibitor (Qiagen, Germany) and centrifuged at 1000 × g for 10 min at 4 °C. The supernatant contains mainly cytosolic proteins and was frozen for western blotting for cytosolic cytochrome c. The pellet was resuspended in an ice-cold disruption buffer (Qiagen, Germany) and centrifuged at 1000 × g for 10 min at 4 °C. The supernatant was transferred to a 1.5-ml tube and centrifuged at 6000 × g for 10 min at 4 °C. The resulting mitochondrial pellet was washed with a mitochondria storage buffer (Qiagen, Germany) and centrifuged at 6000 × g for 20 min at 4 °C. The mitochondrial pellet was resuspended in RIPA lysis buffer (Beyotime, China) and then frozen for western blotting.

Wu W., Zhao D., Shah S.Z., Zhang X., Lai M., Yang D., Wu X., Guan Z., Li J., Zhao H., Li W., Gao H., Zhou X., Qiao J, & Yang L. (2019). OPA1 overexpression ameliorates mitochondrial cristae remodeling, mitochondrial dysfunction, and neuronal apoptosis in prion diseases. Cell Death & Disease, 10(10), 710.

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Protein Immunoprecipitation and Tryptic Digestion

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Samples (40 mg) were resuspended in 800 μL of lysis buffer (0.25% sodium deoxycholate, 1 mM EDTA, 0.5% Igepal in PBS pH 7.4, and protease inhibitor (Roche)), the internal standard was added and the samples were homogenized with a tissue lyser (TissueLyser II, QIAGEN) for 30 min at 30 Hz. After homogenization, lysates were put on rotation at 4°C for 2 h, centrifuged at 21,000 g for 30 min. The supernatants were recovered and incubated with previously blocked protein A magnetic beads (Merck) for 2 h in rotation at 4°C. Beads were recovered and washed with lysis buffer, buffer A (150 mM NaCl, 20 mM Tris in water, pH 7.5) and buffer B (400 mM NaCl, 20 mM Tris in water, pH 7.5). Proteins were eluted with glycine (0.1 M, pH 3) and incubated shaking for 30 min. Cysteines were reduced and carbamylated with TCEP·HCl and IAA respectively. Proteins were finally digested overnight with trypsin at 37°C. Tryptic peptides were purified on C₁₈ Macrospin columns (Harvard Apparatus) according to the manufacturer’s instructions and eluates were dried at room temperature with a vacuum centrifuge (Eppendorf). Dried samples were finally resuspended in a solution of 3% MeCN and 0.1% FA (25 μL). Samples (6 µL) were then injected in the nanoLC-MS system.

Bisbal Lopez L., Ravazza D., Bocci M., Zana A., Principi L., Dakhel Plaza S., Galbiati A., Gilardoni E., Scheuermann J., Neri D., Pignataro L., Gennari C., Cazzamalli S, & Dal Corso A. (2023). Ex vivo mass spectrometry-based biodistribution analysis of an antibody-Resiquimod conjugate bearing a protease-cleavable and acid-labile linker. Frontiers in Pharmacology, 14, 1320524.

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Western Blot Analysis of Apoptosis Markers

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We prepared the RIPA buffer (Beyotime Biotechnology) at a 4°C atmosphere in advance. Once the proteins were isolated from cells, the protease inhibitor (Qiagen, Hilden, Germany) was utilized for protein protection. Those target proteins were transferred into a PVDF membrane (Hyclone) by the way of electrophoresis. The primary antibodies used here consisted of anti-Bcl-2 (ab32124, Abcam, Cambridge, UK), anti-Bax (ab32503), anti-cleaved-caspase 9 (ab2324), anti-matrix metalloproteinase (MMP) 2 (ab37150), anti-cleaved-caspase 3 (ab2302), anti-MMP 9 (ab73734), anti-inhibitor of metalloproteinase 1 (TIMP-1) (ab38978), anti-vimentin (ab92547), anti-mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases (ERK) kinase (MEK) (ab215263), anti-p-MEK (ab194754), anti-p-ERK (ab201015), anti-ERK (ab17942), anti-p-NF-κB-p65 (ab222494), anti-nuclear factor kappa-B (NF-κB)-p65 (ab207297) and anti-β-actin (ab16039). All of them were attached to the target protein bands at 4°C overnight, before the goat anti-rabbit (HRP) (ab7090) was introduced to the membrane at 25°C (1 h). Image Lab™ Software (Bio-Rad) was responsible for protein bands quantification.

Chen Y., Wang Y., Li Y., Li X., Yuan T., Yang S, & Sun X. (2020). The circRNA-MYLK plays oncogenic roles in the Hep-2 cell line by sponging microRNA-145-5p. General physiology and biophysics, 39(3).

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Immunoblotting Analysis of Lipid Metabolism

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These procedures were performed as previously described (Bolsoni-Lopes et al., 2013 (link)). Tissues were dissociated using cell lysis buffer containing protease inhibitors (Qiagen, Hilden, Germany). The protein concentrations were measured using the BCA (Thermo Fisher Scientific, USA) method. The tissue protein extracts were separated on SDS-PAGE gels and electro-transferred onto polyvinylidene fluoride membranes (Bio-Rad, USA). Specific proteins were detected using the following specific antibodies: beta-actin (1:2,000, CST, 3700S), HMGCR (1:1,000, ABCAm, ab174830), NPC1L1 (1:1,000, ABCAm, ab121000), LXRα (1:1,000, ABCAm, Ab176323), CYP7A1 (ABCAm, ab65596). Secondary horseradish peroxidase-conjugated antibodies were used at 1:2,000 dilutions. The proteins were detected using Western Chemiluminescent HRP Substrate (Cell Signaling Technology, USA). All experiments were repeated at least three times.

Huang W.W., Hong B.H., Bai K.K., Tan R., Yang T., Sun J.P., Yi R.Z, & Wu H. (2020). Cis- and Trans-Palmitoleic Acid Isomers Regulate Cholesterol Metabolism in Different Ways. Frontiers in Pharmacology, 11, 602115.

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Expression and Purification of YFP-TDP-43

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Full-length TDP-43 was expressed as a fusion protein with His-tagged Venus YFP at the N-terminus joined by a TEV-cleavable linker. The construct was cloned into the pET-30 vector (EMD Millipore) and expressed in E. coli BL21-AI cells (Life Technologies). Bacterial culture was lysed by sonication on ice with lysis buffer (40 mM HEPES–KOH, pH 7.4, 500 mM KCl, 20 mM MgCl₂, 10 % glycerol, 20 mM imidazole, 2 mM β-mercaptoethanol) containing protease inhibitors (Roche Applied Science). Lysate was centrifuged at 15,000 RPM in a Sorvall SS-34 rotor for 30 min at 4 °C. Prior to Ni–NTA agarose bead column (Qiagen) purification of the lysate supernatant, the column was washed with 5 column volumes of 40 mM HEPES–KOH, pH 7.4, 500 mM KCl, 20 mM MgCl₂, 10 % glycerol, 20 mM imidazole, 2 mM β-mercaptoethanol, and protease inhibitors. The YFP-TDP-43 fusion protein was eluted from the column with 40 mM HEPES, pH 7.4, 500 mM KCl, 20 mM MgCl₂, 250 mM imidazole, 10 % glycerol, 2 mM β-mercaptoethanol, and protease inhibitors. SDS-PAGE analysis of eluent showed that the fusion protein fraction was 95 % pure.

Xiao S., Sanelli T., Chiang H., Sun Y., Chakrabartty A., Keith J., Rogaeva E., Zinman L, & Robertson J. (2015). Low molecular weight species of TDP-43 generated by abnormal splicing form inclusions in amyotrophic lateral sclerosis and result in motor neuron death. Acta Neuropathologica, 130(1), 49-61.

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Purification of Dimeric MmRIPK3 Construct

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The expression of baculovirus construct MmRIPK3 (S02-H303) N- terminal histidine, which is the same construct used for PDB deposition 4M69, carrying the same point mutations (distal to dimer interface), was expressed in T.ni cells. The cell paste was homogenized in 50 mM tris (pH 8.0), 0.5 M NaCl, 5% glycerol, 10 mM imidazole, 10 mM BME, 0.25% CHAPS, and protease inhibitors (Roche), and the lysate was incubated with Ni-NTA beads (Qiagen) for 1 hour at 4°C. The protein bound to Ni-NTA resin was pelleted by centrifugation at 1900 rpm. The resin was then packed into a gravity column and washed with the lysis buffer supplemented with 30 mM imidazole. The bound protein was then eluted using lysis buffer + 250 mM imidazole and immediately injected onto S200 10/300 Increase column (GE Healthcare) that had been preequilibrated with 50 mM tris (pH 8.0), 500 mM NaCl, 5% glycerol, 1 mM tris(2-carboxyethyl)phosphine. Gel filtration standards (Bio-Rad) were then injected over the same column. Ve/Vo was plotted against the log of molecular weight to generate a standard curve. The red triangle indicates the position of MmRIPK3, indicating that the construct is a dimer at the RIPK3 concentration injected (1 mg/ml).

Raju S., Whalen D.M., Mengistu M., Swanson C., Quinn J.G., Taylor S.S., Webster J.D., Newton K, & Shaw A.S. (2018). Kinase domain dimerization drives RIPK3-dependent necroptosis. Science signaling, 11(544), eaar2188.

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Western Blot Analysis of CD, DPD and β-Actin

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Cells were lysed in RIPA buffer (Sigma-Aldrich, St Louis, MI, USA) with protease inhibitors (Qiagen, Hilden, Germany). Proteins (20 µg) were separated by SDS-PAGE gel electrophoresis and transferred to polyvinylidene difluoride membrane (Millipore, Billeroca, MA, USA). The membranes were incubated with antibodies specific to CD (obtained from Dr. Suh-Kim, Aju University, Korea), DPD (Cell Signaling, Beverly, MA, USA), and β-actin (Sigma-Aldrich, St Louis, MO, USA). Membranes were then treated with HRP conjugated anti-rabbit (Cell Signaling, Beverly, MA, USA) or anti-mouse (Invitrogen, Carlsbad, CA, USA) secondary antibody. The specific proteins were visualized by chemo-luminescence (Roche, Indianapolis, IN, USA) according to the manufacturer's protocol and detected with LAS-3000 (Fuji Film, Stockholm, Sweden).

Chung T., Na J., Kim Y.I., Chang D.Y., Kim Y.I., Kim H., Moon H.E., Kang K.W., Lee D.S., Chung J.K., Kim S.S., Suh-Kim H., Paek S.H, & Youn H. (2016). Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas. Theranostics, 6(10), 1477-1490.

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Mitochondria Isolation and Fractionation

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Cells plated into 10 cm petri dishes and confluent 70–80% were washed with cold PBS and then suspended in cold Lysis Buffer (Qproteome Mitochondria Isolation Kit, Qiagen, Venlo, The Netherlands) complemented with protease (Bimake, Houston, TX, USA) and phosphatase inhibitors (Merck KGaA). Cells were incubated in continuous rotation for 10 min at 4 °C and then centrifuged at 1100 g for 10 min at 4 °C to precipitate permeabilized cells. The cell pellet was separated from the supernatant (which contained soluble proteins) and was resuspended in cold disruption Buffer (Qiagen, Venlo, The Netherlands) supplemented with phosphatase and protease inhibitors. The pellet was homogenized with 15–20 repeated aspirations with a blunt-end needle (gauge 26). The suspension was centrifuged at 1100 g for 5 min. The pellet containing the PNS fraction (nuclei, cell debris, and unbroken cells) was discarded. The supernatant was centrifuged at 7000 g for 10 min at 4 °C. The pellet containing the mitochondria-enriched fraction was resuspended in cold supplemented RIPA Buffer 1x (NaCl 150 mM; Tris-HCl 50 mM; NP-40 1%; sodium deoxycholate 0.5%; SDS 0.1%). The supernatant was enriched in the microsomal fraction.

Grisan F., Iannucci L.F., Surdo N.C., Gerbino A., Zanin S., Di Benedetto G., Pozzan T, & Lefkimmiatis K. (2021). PKA compartmentalization links cAMP signaling and autophagy. Cell Death and Differentiation, 28(8), 2436-2449.

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