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Complete protease inhibitor cocktail tablet

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Complete protease inhibitor cocktail tablets are a laboratory product designed to inhibit the activity of various proteases. These tablets provide a broad-spectrum solution for the inhibition of proteolytic enzyme activity in biological samples.

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Protease inhibitor cocktail (10240 citations) Complete protease inhibitor (1691 citations) Protease inhibitor tablet (349 citations) COmplete inhibitor cocktail (34 citations) Complete protease cocktail (22 citations) Proteases inhibitor cocktail (19 citations) Complete Cocktail tablets (9 citations) Complete Protease tablets (5 citations) Complete® inhibitors cocktail (4 citations) Protease cocktail tablet (2 citations)

581 protocols using complete protease inhibitor cocktail tablet

1

Purification of Ada2b Complexes for MudPIT Analysis

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Ada2b complexes were Flag-affinity purified from 12 l of 1e7/l S2 Ada2b-PBH2F2 cells as described before with minor modifications to accommodate MudPIT analysis after gel filtration (31 (link)). The nuclear extract (∼800 mg) was treated with benzonase for 1 h at 4°C, followed by ultracentrifugation for two hours at 50 000 RPM at 4°C. The cleared nuclear extract was frozen and thawed on ice for a three hours affinity purification with M2 mouse-anti Flag conjugated beads (Millipore Sigma) at a ratio of 100 mg extract to 75 μl packed beads. After four high salt batch washes, Ada2b-PB complexes were eluted for one hour in one bead volume of 0.5 mg/ml 3× Flag peptide (Custom order, Penn State Milton S. Hershey Medical Center College of Medicine, NH2-END-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-lys-Gly-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-COOH) in 300 mM NaCl, 1.5 mM MgCl2, 5% glycerol, 0.05% Triton X-100, 20 mM HEPES pH 7.5 and 1 mM PMSF, 0.2% [w/v] leupeptin, 0.2% [w/v] pepstatin A, 1× Roche cOmplete Protease Inhibitor Cocktail Tablets at 4°C.
For HAT assays, the Flag immunoprecipitation was performed overnight, and the complexes were eluted for 1 h in 1.0 mg/ml 3× Flag in 150 mM NaCl, 1.5 mM MgCl2, 5% glycerol, 0.05% Triton X-100, 20 mM HEPES pH 7.5, 1 mM PMSF, 0.2% [w/v] leupeptin, 0.2% [w/v] pepstatin A, and 1× Roche cOmplete Protease Inhibitor Cocktail Tablets at 4°C.
Soffers J.H., Li X., Saraf A., Seidel C.W., Florens L., Washburn M.P., Abmayr S.M, & Workman J.L. (2019). Characterization of a metazoan ADA acetyltransferase complex. Nucleic Acids Research, 47(7), 3383-3394.
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2

Purification of GST and MBP Fusion Proteins

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Plasmids were transformed in BL21 (DE3) Competent E. Coli cells and grown in LB media (GST fusion proteins) or LB media with 0.2% glucose (MBP fusion proteins) with ampicillin. At an OD600 of 0.6–0.8, cells were induced with 0.3 mM IPTG for 4 hours at 37°C. Cells were then harvested by centrifugation (4000 rpm for 20 min) and washed with phosphate-buffered saline (PBS). Cells were lysed by sonication in GST column buffer (40% PBS, 60% TEN buffer: 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, pH 8.0, 100 mM NaCl) supplemented with complete protease inhibitor cocktail tablets (Roche #04693159001) and 10 mg lysozyme for GST fusion proteins or in maltose column buffer (20 mM Tris-HCl, pH 7.4, 0.3 M NaCl, and 1 mM EDTA) with 1mM DTT and complete protease inhibitor cocktail tablets for MBP fusion proteins. Lysates were cleared by centrifugation (15,000 rcf for 20 min) and incubated with glutathione sepharose (BioVision #6566) for GST fusion proteins or amylose resin (New England Biolabs #E8021S) for MBP fusion proteins for 1 hour at 4°C. Beads were poured over a column and washed with column buffer. GST fusion proteins were eluted with 20 mM glutathione in 75 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM DTT, and 0.1% Triton-X. MBP fusion proteins were eluted with 10 mM maltose in maltose column buffer. Proteins were dialyzed overnight into PBS.
Huynh O., Ruis K., Montales K, & Michael W.M. (2023). NBS1 binds directly to TOPBP1 via disparate interactions between the NBS1 BRCT1 domain and the TOPBP1 BRCT1 and BRCT2 domains. DNA repair, 123, 103461.
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3

Purification of GST and MBP Fusion Proteins

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Plasmids were transformed in BL21 (DE3) Competent E. Coli cells and grown in LB media (GST fusion proteins) or LB media with 0.2% glucose (MBP fusion proteins) with ampicillin. At an OD600 of 0.6–0.8, cells were induced with 0.3 mM IPTG for 4 hours at 37 °C. Cells were then harvested by centrifugation (4000 rpm for 20 min) and washed with phosphate-buffered saline (PBS). Cells were lysed by sonication in GST column buffer (40% PBS, 60% TEN buffer: 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, pH 8.0, 100 mM NaCl) supplemented with cOmplete protease inhibitor cocktail tablets (Roche #04693159001) and 10 mg lysozyme for GST fusion proteins or in maltose column buffer (20 mM Tris-HCl, pH 7.4, 0.3 M NaCl, and 1 mM EDTA) with 1mM DTT and cOmplete protease inhibitor cocktail tablets for MBP fusion proteins. Lysates were cleared by centrifugation (15,000 rcf for 20 min) and incubated with glutathione sepharose (BioVision #6566) for GST fusion proteins or amylose resin (New England Biolabs #E8021S) for MBP fusion proteins for 1 hour at 4 °C. Beads were poured over a column and washed with column buffer. GST fusion proteins were eluted with 20 mM glutathione in 75 mM HEPES, pH 7.5, 150 mM NaCl, 5 mM DTT, and 0.1% Triton-X. MBP fusion proteins were eluted with 10 mM maltose in maltose column buffer. Proteins were dialyzed overnight into PBS.
Kim A., Montales K., Ruis K., Senebandith H., Gasparyan H., Cowan Q, & Michael W.M. (2020). Biochemical analysis of TOPBP1 oligomerization. DNA repair, 96, 102973.
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4

Fractionation of Soluble and Chromatin-Associated Proteins

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For fractionation of soluble and chromatin-associated proteins, mESCs were lysed in CSK buffer (0.5% Triton X-100, 10 mM Hepes (pH 7.4), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 5 mM NaF, 1 mM Na3VO4, 1mM sodium pyrophosphate, 1 mM β-glycerophosphate plus 0.1 U/ml Complete Protease Inhibitor Cocktail Tablets (Roche)), and incubated on ice for 5 minutes. Samples were centrifuged at 13000 rpm for 5 mins and supernantant (soluble fraction) saved. The pellet was washed 3 times with CSK buffer and resuspended in NaCl buffer (0.1% Triton X-100, 50mM TRIS (pH 7.4), 250 mM NaCl, 1 mM EDTA, 5 mM NaF, 1 mM Na3VO4, 1 mM sodium pyrophosphate, 1 mM β-glycerophosphate plus 0.1 U/ml Complete Protease Inhibitor Cocktail Tablets (Roche)) with Benzonase (Sigma E1014-5KU) 1:500. Samples were incubated on ice for 30 mins and resuspended every 10 mins. Samples were then centrifuged at 13000 rpm for 15 mins and the supernatant (chromatin fraction) was saved. Soluble fraction was centrifuged again before analysis to remove any chromatin contamination.
Segarra-Fas A., Espejo-Serrano C., Bustos F., Zhou H., Wang F., Toth R., Macartney T., Bach I., Nardocci G, & Findlay G.M. (2022). An RNF12-USP26 amplification loop drives germ cell specification and is disrupted by disease-associated mutations. Science signaling, 15(742), eabm5995.
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5

Fractionation of Soluble and Chromatin-Associated Proteins

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For fractionation of soluble and chromatin-associated proteins, mESCs were lysed in CSK buffer (0.5% Triton X-100, 10 mM Hepes (pH 7.4), 100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 5 mM NaF, 1 mM Na3VO4, 1mM sodium pyrophosphate, 1 mM β-glycerophosphate plus 0.1 U/ml Complete Protease Inhibitor Cocktail Tablets (Roche)), and incubated on ice for 5 minutes. Samples were centrifuged at 13000 rpm for 5 mins and supernantant (soluble fraction) saved. The pellet was washed 3 times with CSK buffer and resuspended in NaCl buffer (0.1% Triton X-100, 50mM TRIS (pH 7.4), 250 mM NaCl, 1 mM EDTA, 5 mM NaF, 1 mM Na3VO4, 1 mM sodium pyrophosphate, 1 mM β-glycerophosphate plus 0.1 U/ml Complete Protease Inhibitor Cocktail Tablets (Roche)) with Benzonase (Sigma E1014-5KU) 1:500. Samples were incubated on ice for 30 mins and resuspended every 10 mins. Samples were then centrifuged at 13000 rpm for 15 mins and the supernatant (chromatin fraction) was saved. Soluble fraction was centrifuged again before analysis to remove any chromatin contamination.
Segarra-Fas A., Espejo-Serrano C., Bustos F., Zhou H., Wang F., Toth R., Macartney T., Bach I., Nardocci G, & Findlay G.M. (2022). An RNF12-USP26 amplification loop drives germ cell specification and is disrupted by disease-associated mutations. Science signaling, 15(742), eabm5995.
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6

Investigating MLH1 Protein Solubility

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For analyses of wild-type MLH1, HCT116 cells were transfected as described above. Cells were harvested in buffer B (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 1 mM EDTA) supplemented with complete protease inhibitor cocktail tablets (Roche), and lysed by sonication (3 × 10 s). Then the lysate was distributed into different tubes that were incubated at 30 min. at the indicated temperatures. The soluble and insoluble fractions were separated by centrifugation (15000 g, 4°C, 30 min), after which the supernatant was removed and the pellet washed once in buffer B. Subsequently, SDS sample buffer was added, and the final sample volume was kept identical between the pellet and the supernatant. Finally, fractions were analyzed by SDS-PAGE and western blotting as described.
For comparison of the variants, HCT116 cells were transfected and treated with bortezomib as described above. Cells were harvested in buffer B supplemented with complete protease inhibitor cocktail tablets (Roche), and lysed by sonication (3 × 10 s). The soluble and insoluble fractions were then immediately separated by centrifugation and analyzed SDS-PAGE and western blotting as described above.
Abildgaard A.B., Stein A., Nielsen S.V., Schultz-Knudsen K., Papaleo E., Shrikhande A., Hoffmann E.R., Bernstein I., Gerdes A.M., Takahashi M., Ishioka C., Lindorff-Larsen K, & Hartmann-Petersen R. (2019). Computational and cellular studies reveal structural destabilization and degradation of MLH1 variants in Lynch syndrome. eLife, 8, e49138.
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7

Tissue and Blood Sample Collection

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Blood was collected from cardiac puncture and immediately centrifuged (138× g, 10 min, 4 °C) to separate plasma from erythrocytes. Plasma was collected and stored in aliquots at −80 °C, until further analysis, while red blood cells were hemolyzed with distilled water (1:1 v/v). Subsequently, the samples were centrifuged (400× g, 15 min, 4 °C) and the supernatant (i.e., red blood cell lysate, RBCL) was collected and stored in aliquots at −80 °C. Liver, heart, left kidney, right kidney, and thyroid gland were excised, snapped-frozen in liquid nitrogen, and kept at −80 °C, until further analysis. For homogenization, the tissues were weighed and mixed with phosphate-buffered saline (PBS) containing a Roche cOmplete™ protease inhibitor cocktail tablet (Roche Diagnostics, Mannheim, Germany). The samples were transferred to Minilys personal homogenizer (Bertin Instruments, Montigny-le-Bretonneux, France), homogenized for 30 s at 5000 rpm, placed on ice for 20 s, and centrifuged (15,00× g, 5 min, 5 °C). The resulting supernatant (i.e., the tissue homogenate) was collected, divided into aliquots, and stored at −80 °C.
Vardakas P., Veskoukis A.S., Rossiou D., Gournikis C., Kapetanopoulou T., Karzi V., Docea A.O., Tsatsakis A, & Kouretas D. (2022). A Mixture of Endocrine Disruptors and the Pesticide Roundup® Induce Oxidative Stress in Rabbit Liver When Administered under the Long-Term Low-Dose Regimen: Reinforcing the Notion of Real-Life Risk Simulation. Toxics, 10(4), 190.
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8

Biotin-phenol Labeling for Proteomics

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Biotin-phenol labeling in live cells was performed according to previously published protocols.41 (link), 45 (link) Briefly, constructs harboring the MIEF1-MP–APEX fusion proteins or APEX (control sample) was transfected into HEK293T cells using Lipofectamine 2000. Twenty-four hours post-transfection, cell culture medium was changed to fresh growth medium containing 500 μM biotin-tyramide (CDX-B0270, Adipogen). After incubation at 37 °C for 30 min, H2O2 was added to each plate at a final concentration of 1 mM, and the plates were gently agitated for 1 min. Cells were then washed three times with a quenching solution (5 mM Trolox, 10 mM sodium azide, and 10 mM sodium ascorbate in PBS), and pelleted by centrifugation at 1000g for 5 min. Cell pellets were lysed on ice for 20 min in RIPA buffer (Thermo catalog no. 89901) supplemented with a Roche complete protease inhibitor cocktail tablet and 1 mM PMSF followed by centrifugation at 20,000g for 20 min at 4 °C to remove cell debris. Cell lysates were added to prewashed streptavidin agarose resin (ThermoFisher, # 20359), rotated at 4 °C overnight, and then washed three times with TBST and 0.5% (v/v) sodium dodecyl sulfate (SDS) at room temperature. Bound proteins were eluted with 2×SDS by boiling at 95 °C and analyzed by proteomics and Western blotting.
Rathore A., Chu Q., Tan D., Martinez T.F., Donaldson C.J., Diedrich J.K., Yates JR I.I.I, & Saghatelian A. (2018). MIEF1 Microprotein Regulates Mitochondrial Translation. Biochemistry, 57(38), 5564-5575.
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9

FLAG-Tagged Microprotein Purification

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FLAG-tagged microprotein constructs
[or the empty pcDNA3.1(+) vector] were transfected into a 10 cm dish
of HEK293T cells using Lipofectamine 2000 according to the manufacturer’s
protocol. Forty-eight hours post-transfection, cells were harvested
and lysed in RIPA buffer (Thermo catalog no. 89901) supplemented with
a Roche complete protease inhibitor cocktail tablet and 1 mM PMSF.
Cells were lysed on ice for 20 min followed by centrifugation at 20000g for 20 min at 4 °C to remove cell debris. Cell lysates
were added to prewashed mouse IgG agarose beads (Sigma catalog no.
A0919) and rotated at 4 °C for 1 h. The supernatants were collected
and added to prewashed anti-FLAG M2 Affinity Gel (Sigma, catalog no.
A2220). The suspensions were rotated at 4 °C overnight and washed
four times with TBST. Bound proteins were eluted with 3× FLAG
peptide (Sigma, catalog no. F4799) at 4 °C for 1 h. The eluents
were then separated by sodium dodecyl sulfate–polyacrylamide
gel electrophoresis (SDS–PAGE) and analyzed by Western blotting
using the indicated antibodies.
Chu Q., Rathore A., Diedrich J.K., Donaldson C.J., Yates JR I.I.I, & Saghatelian A. (2017). Identification of Microprotein–Protein Interactions via APEX Tagging. Biochemistry, 56(26), 3299-3306.
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10

Immunoblotting of YFP-FKBP Fusion Proteins

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A 293 T cells were transfected with the YFP‐FKBP‐tagged probes. 48 h after transfection, cells were lysed using RIPA lysis buffer [Tris–HCl 50 mM, EGTA 20 mM, NaCl 9%, Triton X‐100 1%, Roche complete protease inhibitor cocktail tablet (Roche, 04719964001)]. The post‐nucleus lysates were analyzed by SDS–PAGE, followed by immunoblotting with anti‐Venus antibody (1:1,000, MyBioSource, MBS448126) and horseradish peroxidase‐conjugated anti‐Goat antibody. The blotting results were visualized by Amersham™ ECL Select™ (FE Healthcare, RPN2235) and imaged by iBright™ FL1500 Instrument (Thermo Scientific).
Cheng H., Kao Y., Chen T., Sharma L., Yang W., Chuang Y., Huang S., Lin H., Huang Y., Kao C., Yang L., Bearon R., Cheng H., Hsia K, & Lin Y. (2022). Actin filaments form a size‐dependent diffusion barrier around centrosomes. EMBO Reports, 24(1), e54935.
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