Protease inhibitor tablet

Manufactured by Roche

Sourced in United States, Germany, Switzerland, United Kingdom, Canada, China

Protease inhibitor tablet is a laboratory equipment used in the analysis and study of proteins. It functions to inhibit the activity of proteases, which are enzymes that break down proteins. This product helps maintain the integrity of protein samples during experimental procedures.

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Lab products found in correlation

349 protocols using protease inhibitor tablet

Nuclear Protein Extraction Protocol

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The cells
were collected by centrifugation and washed with cold PBS. The pellet
was then resuspended in 5 times the packed cell volume of lysis buffer
(10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, pH 7.9) supplemented
with a protease inhibitor tablet (Roche). The cells were allowed to
swell on ice for 20 min, then centrifuged, and the supernatant was
removed. The pellet was resuspended in 2 times the packed cell volume
and then dounced 10 times with a glass dounce homogenizer. The homogenate
was centrifuged and the supernatant was removed, repeating several
times with fresh lysis buffer. The nuclear pellet was frozen and stored
at −80 °C until use. After thawing, the pellet was resuspended
in 0.9 times the packed nuclear volume in the second lysis buffer
(20 mM HEPES, 0.4 M NaCl, 1.5 mM MgCl₂, 20% glycerol (v/v),
pH 7.9) supplemented with a protease inhibitor tablet (Roche). The
suspension was dounced 20 times with the glass dounce homogenizer.
After incubation at 4 °C for 30 min on a rotator, the sample
was centrifuged at high speed (12 000 rpm) for 30 min. The
supernatant was removed and then dialyzed into a glycerol free, lower
salt buffer (20 mM HEPES, 100 mM KCl, 2 mM MgCl₂, pH 7.9)
supplemented with a protease inhibitor tablet (Roche). The lysate
was aliquoted, flash frozen in liquid nitrogen, and stored at −80
°C until needed.

Marholz L.J., Chang L., Old W.M, & Wang X. (2014). Development of Substrate-Selective Probes for Affinity Pulldown of Histone Demethylases. ACS Chemical Biology, 10(1), 129-137.

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Flag-Tagged Protein Immunoprecipitation

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For each sample, 100µl of fly heads/~10 7 S2 cells were homogenized in 500µl/1ml lysis buffer (50mM TRIS HCL 7.4, 150mM NaCl, 1mM EDTA, 1% TRITON X100 and protease inhibitor tablets [Roche]). Homogenate was kept on ice for 30 min and then sonicated using a Bioruptor sonication device (Diagenode) for 2 min (10 sec on 10 sec off). Sonicated lysate was centrifuged at 21,000g for 10 min, and the supernatant was then collected; 35µl were removed for input. Pre-clean: protein A/G PLUS-Agarose beads (Santa Cruz) were washed 3 times with 150µl of lysis buffer, resuspended with 100µl lysis buffer, and then added to the lysate. The samples were rotated for 30 min at 4°C and centrifuged at 3000 rpm for 1 min. The lysate was collected to a new tube and the beads were discarded. IP: 25µl of Red ANTI-FLAG M2 Affinity Gel from SIGMA ALDRICH (F2426) were washed 3 times with TBS (50mM TRIS pH 7.5,150mM NaCl and protease inhibitor tablets [Roche]) and then added to samples. The samples were rotated for 3 hours at 4°C. Next, the samples were centrifuged at 3000 rpm for 1 min and the unbound lysate was collected to a new tube. Beads were washed 3 times using 400µl washing buffer (300mM NaCl, 0.1% NP 40,50mM TRIS HC1 7.5 and protease inhibitor tablets [Roche]) and twice using 400µl of TBS. For western blot, the beads were eluted with SDS-PAGE X1 sample buffer and boiled at 95°C for 5 min.

Herman N., Kadener S., & Shifman S. (2021). The chromatin factor ROW cooperates with BEAF-32 in regulating long-range inducible genes.

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Proteomics of Saccharomyces cerevisiae and Cell Lines

Cited 2 times

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Saccharomyces cerevisae (BY4742) was grown in 500 mL YPD cultures to an OD600 of 0.8 then washed twice with ice-cold PBS, pelleted, and stored at −80 °C until use. Cells were resuspended in lysis buffer (8 M urea, 50 mM EPPS pH 8.5, 150 mM NaCl, Roche protease inhibitor tablet) and lysed by bead beating. After lysis and bead removal, the lysate was centrifuged to remove cellular debris and the supernatant was collected for use. Cell lines were grown to confluence in DMEM containing 10% fetal bovine serum and 1% streptomycin/puromycin. Cells were harvested by manual scraping and washed twice with PBS. Cells were syringe lysed in lysis buffer (8 M urea, 50 mM EPPS pH 8.5, 150 mM NaCl, and Roche protease inhibitor tablet) and the resulting lysates were cleared via centrifugation.
Desired protein amounts were aliquoted and chloroform methanol precipitated, followed by digestion with LysC (overnight at room temperature, vortex speed 2; Wako) and trypsin (6 h, 37 °C; Promega) digestion. Peptides were labeled with TMT reagents as described previously.^{6 (link),13 (link)} Labeled peptides were mixed, and dried to remove organic solvent prior to cleanup via Sep-Pak (50 mg C₁₈ SepPak; Waters). As needed, labeled peptide mixtures were separated via high-pH reversed phase chromatography and pooled into fractions.^{13 (link)} Samples were dried and stored at −80 °C prior to analysis.

Schweppe D.K., Eng J.K., Yu Q., Bailey D., Rad R., Navarrete-Perea J., Huttlin E.L., Erickson B.K., Paulo J.A, & Gygi S.P. (2020). Full-Featured, Real-Time Database Searching Platform Enables Fast and Accurate Multiplexed Quantitative Proteomics. Journal of proteome research, 19(5), 2026-2034.

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Cellular Fractionation and Nuclear Isolation

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Approximately 1.5 x 10 7 cells were harvested by centrifuging at 1200rpm for 5 minutes at room temperature and washed in PBS. As much supernatant was removed from the pellet as possible, which was then re-suspended in 150ul cold Buffer RLN and incubated on ice for 10 minutes. One protease inhibitor tablet (Roche, Cat. 04693159001) was added to 10ml Buffer RLN immediately before use. The mix was centrifuged at 3700 rpm for 5 minutes at 4 o C and 100ul supernatant was transferred to a new eppendorf (cytosolic fraction). The remainder of the supernatant was discarded and the pellet was washed twice with 100ul Buffer RLN, centrifuging between washes as before. The resulting pellet was re-suspended in ~80ul lysis buffer.
One protease inhibitor tablet (Roche, Cat. 04693159001) was added to 10ml lysis buffer immediately before use. Samples were then homogenized using a 1ml syringe and needle (21G x 1.5" -Nr.2., 0.8mm x 40mm) to lyse nuclei, and incubated on ice for 30 minutes before centrifuging at 14,000rpm for 20 minutes at 4 o C. The resulting supernatant was pipetted into a fresh, sterile eppendorf (nuclear fraction) and stored at -20 o C.

Jones R., Wijesinghe S.N., Halsall J.A., & Kanhere A. (2020). A long intergenic non-coding RNA regulates nuclear localisation of DNA methyl transferase-1.

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FLAG-tagged Protein Immunoprecipitation

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Cells were lysed with lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with protease inhibitor tablet (Roche) and rotated for 30 minutes at 4°C. The cell lysate was then spun for 10 minutes at 15,000 rpm, 4°C. For cell fractionations, we performed REAP fractionation as described previously [38 (link)]. For each immunoprecipitation, 2.5 mg of protein was mixed with 20 μL of anti-FLAG M2 magnetic bead slurry (Sigma) in a 1 mL volume, using lysis buffer without detergent to fill the volume. The mixture was rotated overnight at 4°C and subsequently washed three times with wash buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% NP-40) supplemented with protease inhibitor tablet (Roche), with 5 minutes of rotation at 4°C per wash. The beads were resuspended in 25 μL 2X Laemmli buffer and boiled to elute immunoprecipitants (IP). For western blot analysis, 1% input and 100% of IP were used for SDS-PAGE and probed with anti-FLAG antibody (1:3000, Sigma F7425).

Ly M., Burgess H.M., Shah S.B., Mohr I, & Glaunsinger B.A. (2022). Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing. PLoS Pathogens, 18(2), e1010099.

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Co-immunoprecipitation of protein complexes

Cited 1 time

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Co-immunoprecipitation (CoIP) was performed as previously described (58 (link)), with modifications. Three days after infection, cells were scraped in ice-cold PBS and lysed in 800 μl of lysis buffer [50 mM tris-HCl at pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% sodium deoxycholate, and 2× protease inhibitor tablet (Roche)] for 30 min on ice and cleared by centrifugation at 14,000g for 10 min at 4°C. Lysates (50 μl) were saved for “Input”. For each pull-down, 2 μg of Flag M2 antibody (Sigma-Aldrich) was bound to 20 μl of washed Dynabeads Protein G (Invitrogen). Lysates were then added to antibody-coupled beads. Reactions were incubated on a rotating platform for 3 hour at 4°C, and then beads were washed five times with 1 ml of wash buffer [50 mM tris-HCl at pH 7.5, 150 mM NaCl, 0.05% NP-40, and 2× protease inhibitor tablet (Roche)]. Immunoprecipitated proteins were eluted with 40 μl of elution buffer [36 μl of lysis buffer supplemented with 4 μl of 3× Flag peptide at 10 mg/ml (Sigma-Aldrich)] with shaking at 1000 rpm for 30 min at room temperature. Immunoblotting for Input and CoIP samples was performed as described earlier.

Shah A.M., Guo L., Morales M.G., Jaichander P., Chen K., Huang H., Cano Hernandez K., Xu L., Bassel-Duby R., Olson E.N, & Liu N. (2023). TWIST2-mediated chromatin remodeling promotes fusion-negative rhabdomyosarcoma. Science Advances, 9(17), eade8184.

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Immunoprecipitation of FLAG-tagged Proteins

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Cells were lysed with lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5% NP-40) supplemented with protease inhibitor tablet (Roche) and rotated for 30 minutes at 4°C. The cell lysate was then spun for 10 minutes at 15,000 rpm, 4°C. For each immunoprecipitation, 2.5 mg of protein was mixed with 20 μL of anti-FLAG M2 magnetic bead slurry (Sigma) in a 1 mL volume, using lysis buffer without detergent to fill the volume. The mixture was rotated overnight at 4°C and subsequently washed three times with wash buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.05% NP-40) supplemented with protease inhibitor tablet (Roche), with 5 minutes of rotation at 4°C per wash. The beads were resuspended in 25 μL 2X Laemmli buffer and boiled to elute immunoprecipitants (IP). For western blot analysis, 1% input and 100% of IP were used for SDS-PAGE and probed with anti-FLAG antibody (1:3000, Sigma F7425).

Ly M., Burgess H.M., Mohr I., & Glaunsinger B.A. (2021). Vaccinia virus D10 has broad decapping activity that is regulated by mRNA splicing.

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Isolation of Mitochondria from Mouse Skeletal Muscle

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Mitochondria were isolated from mouse skeletal muscle by homogenization using a polytron followed by a Douncer homogenizer with a Teflon pestle in homogenization buffer I (0.1 M KCl, 5 mM MgCl2, 5 mM EGTA, 5 mM sodium pyrophosphate, pH 6.8, and protease inhibitor tablet, Roche). Homogenates were centrifuged at 1,300×g for 10 min at 4 °C. The supernatant (SN1) was kept, and the pellet was resuspended in buffer II (0.25 M sucrose, 50 mM KCL, 5 mM EDTA, 1 mM sodium pyrophosphate, pH 7.4, and protease inhibitor tablet, Roche) and centrifuged at 1,300×g for 10 min at 4 °C. The supernatant (SN2), together with SN1 were centrifuged at 9,000×g for 15 min at 4 °C. The pellet (isolated mitochondria) was resuspended in buffer II.

López-Soldado I., Torres A.G., Ventura R., Martínez-Ruiz I., Díaz-Ramos A., Planet E., Cooper D., Pazderska A., Wanic K., O'Hanlon D., O'Gorman D.J., Carbonell T., de Pouplana L.R., Nolan J.J., Zorzano A, & Hernández-Alvarez M.I. (2023). Decreased expression of mitochondrial aminoacyl-tRNA synthetases causes downregulation of OXPHOS subunits in type 2 diabetic muscle. Redox Biology, 61, 102630.

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Subcellular Fractionation of T Cells

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To isolate nuclear and cytoplasmic fractions (53 (link)), ten million T cells were incubated in 200 μl of hypotonic buffer (10 mM Hepes pH 7.9, 10 mM KCl, 0.1 mM EDTA, 5 mM MgCl₂) supplemented by 1 mM DTT and a protease inhibitor tablet (Roche) for 30 min. Swollen cells were lysed with 0.1% Triton X-100 and vortexed for 20s, before being centrifuged at 13,000g for 10 min at 4 °C. The cytoplasmic fraction was carefully removed to another tube, and an equal volume of 2× SDS sampling buffer was added. The nuclear pellet was resuspended in 200 μl of hypertonic buffer (20 mM Hepes pH7.9, 0.4 M NaCl, 1 mM EDTA) supplemented with 1 mM DTT, and a protease inhibitor tablet (Roche), and incubated for 30 min. An equal volume of 2× SDS sampling buffer was then added to the resuspended nuclear fraction.

Li H., Hostager B.S., Arkee T, & Bishop G.A. (2021). Multiple mechanisms for TRAF3-mediated regulation of the T cell costimulatory receptor GITR. The Journal of Biological Chemistry, 297(3), 101097.

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DENV-Induced Host Protein Interactome

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Huh7 cells were infected with DENV at a multiplicity of infection (MOI) of ten. Two hours before cell harvest, cell media was spiked with 440μCi of [³⁵S]-methionine and [³⁵S]-cysteine (Easy tag EXPRESS³⁵S; Perkin Elmer; Waltham, MA). Cells were lysed in lysis buffer (0.1% Triton; 150mM NaCl; 60mM MgCl₂; 25mM Tris-HCl, pH 7.5; 1mM DTT, 1μM Microcystin (Cayman Chemical; Ann Arbor, MI), and 1× protease inhibitor tablet (Roche; Mannheim, Germany)), clarified by centrifugation, and incubated with a γ-phosphate ATP-affinity resin. The resin was washed 3× with low salt wash buffer (50mM Tris, pH 7.5; 150mM NaCl; and 60mM Mg Cl₂) and bound proteins were removed from the resin by boiling in 5× SDS running buffer. Samples were subjected to SDS-PAGE and analyzed by silver stain. The gel was then dried and analyzed by autoradiogram for seven days. Indicated bands were excised and identified by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS) on an ABSCIEX (Framingham, MA) TOF/TOF 5800 mass spectrometer.

Howe M.K., Speer B.L., Hughes P.F., Loiselle D.R., Vasudevan S, & Haystead T.A. (2016). An Inducible Heat Shock Protein 70 Small Molecule Inhibitor Demonstrates Anti-Dengue Virus Activity, Validating Hsp70 as a Host Antiviral Target. Antiviral research, 130, 81-92.

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