Protease inhibitor cocktail, by Merck Group - Product details

1

Co-Immunoprecipitation Protocols for Endogenous and Overexpressed Proteins

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For the Co-Immunoprecipitation of endogenous protein, the cells were lysed in IP lysis buffer with 1xProtease Inhibitor Cocktail (Sigma) and 10 mM NaF, and then the cell lysates were centrifuged for 10 min, 2,500 g at 4 °C. The cleared cell lysates were incubated with primary antibody against sheep anti-STING or rabbit anti-STEEP overnight at 4°C. Dynabeads™ Protein G (Invitrogen) was added into the mixtures for extra 2 h. After 4-6 times wash with IP lysis buffer, the immunoprecipitated complexes were eluted by glycine buffer (200 mM glycine, pH 2.5) with Protease Inhibitor Cocktail (Sigma) and 10 mM NaF, and the elutes were neutralized with 1M Tris-Hcl pH 10.4 and then evaluated by Immunoblotting.
For the Co-Immunoprecipitation of overexpressed proteins, the HEK-293T cells were transfected for 24 h by lipofectamine 2000, and then were lysed by IP lysis buffer with 1xProtease Inhibitor Cocktail and 10 mM NaF. After centrifugation at 14000 g for 10 min at 4 °C, the cleared cell lysates were incubated with FLAG® M2 Magnetic Beads (Sigma) for 2 h at RT. After washing 4 times with 1xTBS buffer with 0.05% tween 20 (TBST), the immunoprecipitated complexes were eluted by 3X FLAG® tag peptide (Sigma-Aldrich) with Protease Inhibitor Cocktail (Sigma) and 10mM NaF, and the elutes were evaluated by Immunoblotting.

Zhang B.C., Nandakumar R., Reinert L.S., Huang J., Laustsen A., Gao Z.L., Sun C.L., Jensen S.B., Troldborg A., Assil S., Berthelsen M.F., Scavenius C., Zhang Y., Windross S.J., Olagnier D., Prabakaran T., Bodda C., Narita R., Cai Y., Zhang C.G., Stenmark H., Doucet C.M., Noda T., Guo Z., Goldbach-Mansky R., Hartmann R., Chen Z.J., Enghild J.J., Bak R.O., Thomsen M.K, & Paludan S.R. (2020). STEEP mediates STING ER exit and activation of signaling. Nature immunology, 21(8), 868-879.

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Co-Immunoprecipitation Protocols for Endogenous and Overexpressed Proteins

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For the Co-Immunoprecipitation of endogenous protein, the cells were lysed in IP lysis buffer with 1xProtease Inhibitor Cocktail (Sigma) and 10 mM NaF, and then the cell lysates were centrifuged for 10 min, 2,500 g at 4 °C. The cleared cell lysates were incubated with primary antibody against sheep anti-STING or rabbit anti-STEEP overnight at 4°C. Dynabeads™ Protein G (Invitrogen) was added into the mixtures for extra 2 h. After 4-6 times wash with IP lysis buffer, the immunoprecipitated complexes were eluted by glycine buffer (200 mM glycine, pH 2.5) with Protease Inhibitor Cocktail (Sigma) and 10 mM NaF, and the elutes were neutralized with 1M Tris-Hcl pH 10.4 and then evaluated by Immunoblotting.
For the Co-Immunoprecipitation of overexpressed proteins, the HEK-293T cells were transfected for 24 h by lipofectamine 2000, and then were lysed by IP lysis buffer with 1xProtease Inhibitor Cocktail and 10 mM NaF. After centrifugation at 14000 g for 10 min at 4 °C, the cleared cell lysates were incubated with FLAG® M2 Magnetic Beads (Sigma) for 2 h at RT. After washing 4 times with 1xTBS buffer with 0.05% tween 20 (TBST), the immunoprecipitated complexes were eluted by 3X FLAG® tag peptide (Sigma-Aldrich) with Protease Inhibitor Cocktail (Sigma) and 10mM NaF, and the elutes were evaluated by Immunoblotting.

Zhang B.C., Nandakumar R., Reinert L.S., Huang J., Laustsen A., Gao Z.L., Sun C.L., Jensen S.B., Troldborg A., Assil S., Berthelsen M.F., Scavenius C., Zhang Y., Windross S.J., Olagnier D., Prabakaran T., Bodda C., Narita R., Cai Y., Zhang C.G., Stenmark H., Doucet C.M., Noda T., Guo Z., Goldbach-Mansky R., Hartmann R., Chen Z.J., Enghild J.J., Bak R.O., Thomsen M.K, & Paludan S.R. (2020). STEEP mediates STING ER exit and activation of signaling. Nature immunology, 21(8), 868-879.

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Fluorescence-Activated Nuclei Sorting of OPCs

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Fluorescence-activated nuclei sorting (FANS) was used to purify Sun1-GFP⁺ OPC nuclei from brain tissue^{55 (link),91 (link)}. Control or Atrx^Sox10Cre forebrain tissue was homogenized in 500 μL homogenization buffer 20 mM Tricine KOH, 25 mM MgCl₂, 250 mM sucrose, 1 mM DTT, 0.15 mM spermine, 0.5 mM spermidine, 0.1% IGEPAL-630, 1× protease inhibitor cocktail (Millipore Sigma Cat# 11873580001), 1 μL/mL RNase inhibitor (Thermo Fisher Scientific Cat# 10777019). Samples were diluted to 7.5 mL with homogenization buffer and filtered through a 40 µm strainer. Filtered samples were layered on top of 7.5 mL cushion buffer consisting of 0.5 mM MgCl_2, 0.88 M sucrose, 0.5 mM DTT, 1× protease inhibitor cocktail (Millipore Sigma Cat# 11873580001), 1 μL/mL RNase inhibitor (Thermo Fisher Scientific Cat# 10777019) and centrifuged at 2800 g for 20 mins at 4 °C. Nuclei were collected as a pellet, incubated for 10 min in 500 μL 4% FBS, 0.15 mM spermine, 0.5 mM spermidine, 1× protease inhibitor cocktail (Millipore Sigma Cat# 11873580001) and 1 μL/mL RNase inhibitor (Thermo Fisher Scientific Cat# 10777019) in PBS and resuspended by gentle pipetting. Nuclei were sorted using a Sony SH800 Cell Sorter and Sun1GFP⁺ nuclei were collected. Total RNA was immediately isolated from GFP+ nuclei with a single cell RNA purification kit (NorgenBiotek Cat# 51800).

Rowland M.E., Jiang Y., Shafiq S., Ghahramani A., Pena-Ortiz M.A., Dumeaux V, & Bérubé N.G. (2023). Systemic and intrinsic functions of ATRX in glial cell fate and CNS myelination in male mice. Nature Communications, 14, 7090.

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Protein Extraction and Interaction Analysis

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HEK293T cells were lysed in Triton X-100 buffer (150 mM NaCl, 5 mM EDTA, 1% [v/v] Triton X-100, 50 mM HEPES pH 7.4, and protease inhibitor cocktail [Sigma]), NP-40 buffer (150 mM NaCl, 1% [v/v] NP-40, 50 mM HEPES pH 7.4, and protease inhibitor cocktail [Sigma]), or RIPA buffer (150 mM NaCl, 0.5 – 1% [v/v] NP-40, 1% [w/v] deoxycholic acid (DOC), 0.01% [v/v] SDS, 20 mM Tris pH 8.0, and protease inhibitor cocktail [Sigma]), and cell debris pelleted by centrifugation at 13,000 rpm for 20 min at 4 °C. FLAG pull-downs, Co-IP, and western blot analyses were performed as previously described^{42 (link)}. Elution of precipitated proteins was performed by heating samples in Laemmli SDS sample buffer at 95 °C for 5 min.

Sparrer K.M., Gableske S., Zurenski M.A., Parker Z.M., Full F., Baumgart G.J., Kato J., Pacheco-Rodriguez G., Liang C., Pornillos O., Moss J., Vaughan M, & Gack M.U. (2017). TRIM23 mediates virus-induced autophagy via activation of TBK1. Nature microbiology, 2(11), 1543-1557.

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Immunoprecipitation and Immunoblotting of Nuclear Proteins

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Cells were lysed in TNN [50 mM Tris (pH 7.5), 120 mM NaCl, 5 mM EDTA, 0.5% NP40, 10 mM Na₄P₂O₇, 2 mM Na₃VO₄, 100 mM NaF, 1 mM PMSF, 1 mM DTT, 10 mM ß-glycerophosphate, protease inhibitor cocktail (Sigma)]. Proteins were separated by SDS-PAGE, transferred to PVDF membrane and detected by immunoblotting.
For co-immunoprecipitation of endogenous proteins from nuclear extracts, cells were first lysed in [10 mM HEPES pH 7.4, 10 mM NaCl, 3 mM MgCl₂, protease inhibitor cocktail (Sigma)] for 20 min on ice. Pelleted nuclei were resuspended in nuclei lysis buffer [20 mM HEPES pH 7.4, 400 mM NaCl, 1.5 mM MgCl₂, 0.1 mM EDTA, 15% glycerol, 0.5 mM DTT, protease inhibitor cocktail (Sigma)] mixed 1:1 with TNN buffer for another 20 min on ice. After spinning at full speed for 10 min, the supernatant was mixed with 20 mM HEPES pH 7.4 and used for immunoprecipitation overnight at 4°C in constant rotation. Complexes were collected with protein G-dynabeads (Thermo Fisher Scientific) for 2 h at 4°C. Beads were washed three times with nuclei lysis buffer mixed 1:1 with 20 mM HEPES pH 7.4. Proteins were eluted from beads and bound proteins were detected by immunoblotting. Antibodies are listed in Supplementary Table S4.

Fetiva M.C., Liss F., Gertzmann D., Thomas J., Gantert B., Vogl M., Sira N., Weinstock G., Kneitz S., Ade C.P, & Gaubatz S. (2023). Oncogenic YAP mediates changes in chromatin accessibility and activity that drive cell cycle gene expression and cell migration. Nucleic Acids Research, 51(9), 4266-4283.

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Cytosol and Nucleus Protein Fractionation

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For cytosol/nucleus protein fractionation, pelleted cells were resuspended in 3 volumes with respect to the cell pellet of hypotonic buffer (20 mM HEPES pH 7.4, 5 mM NaF, 10 μM NaMoO₄, 0.1 mM EDTA) supplemented with 1× Protease Inhibitor Cocktail (Sigma-Aldrich, Milan, Italy), 1 mM DTT and 1 mM PMSF; upon incubation on ice for 15 min, 0.5% Triton X-100 was added, and lysate was centrifuged at 15,000× g for 30 s at 4 °C in order to collect the cytosolic fraction, which was further clarified by centrifugation at 15,000× g for 15 min. The nuclear pellets were first stratified in sucrose gradient to remove cytosolic contaminants and then resuspended in 1 volume of nuclear lysis buffer (20 mM HEPES, pH 7.4, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA) supplemented with 1× Protease Inhibitor Cocktail (Sigma-Aldrich, Milan, Italy), 1 mM DTT, and 1 mM PMSF; nuclei were incubated for 30 min at 4 °C with gentle shaking and then centrifuged at 15,000× g for 30 min at 4 °C. Salt concentration was adjusted with 1 volume of nuclear lysis buffer without salt addition, supplemented with 1× Protease Inhibitor Cocktail (Sigma-Aldrich, Milan, Italy), 1 mM DTT, and 1 mM PMSF.

Sellitto A., Geles K., D’Agostino Y., Conte M., Alexandrova E., Rocco D., Nassa G., Giurato G., Tarallo R., Weisz A, & Rizzo F. (2019). Molecular and Functional Characterization of the Somatic PIWIL1/piRNA Pathway in Colorectal Cancer Cells. Cells, 8(11), 1390.

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Protein Extraction and Interaction Analysis

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HEK293T cells were lysed in Triton X-100 buffer (150 mM NaCl, 5 mM EDTA, 1% [v/v] Triton X-100, 50 mM HEPES pH 7.4, and protease inhibitor cocktail [Sigma]), NP-40 buffer (150 mM NaCl, 1% [v/v] NP-40, 50 mM HEPES pH 7.4, and protease inhibitor cocktail [Sigma]), or RIPA buffer (150 mM NaCl, 0.5 – 1% [v/v] NP-40, 1% [w/v] deoxycholic acid (DOC), 0.01% [v/v] SDS, 20 mM Tris pH 8.0, and protease inhibitor cocktail [Sigma]), and cell debris pelleted by centrifugation at 13,000 rpm for 20 min at 4 °C. FLAG pull-downs, Co-IP, and western blot analyses were performed as previously described^{42 (link)}. Elution of precipitated proteins was performed by heating samples in Laemmli SDS sample buffer at 95 °C for 5 min.

Sparrer K.M., Gableske S., Zurenski M.A., Parker Z.M., Full F., Baumgart G.J., Kato J., Pacheco-Rodriguez G., Liang C., Pornillos O., Moss J., Vaughan M, & Gack M.U. (2017). TRIM23 mediates virus-induced autophagy via activation of TBK1. Nature microbiology, 2(11), 1543-1557.

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Subcellular Protein Fractionation and Analysis

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Whole protein extracts from mouse tissues and human fibroblast cells were resuspended with lysis buffer (Tris HCl pH 8,0, 25 μM, NaCl 55 μM, EDTA 1 μM, Protease Inhibitor Cocktail, Sigma- Aldrich) and protein concentration was estimated by the Bradford Protein Assay (Thermo Scientific). Proteins, 20–60 μg per lane, were separated by SDS-PAGE.
Subcellular fractionation was performed as previously described, with minor modification55 (link). Briefly, samples were resuspended in 500 μl of buffer A (250 mM sucrose, 50 mM Tris–HCl, pH 7.4, 5 mM MgCl2, Protease Inhibitor Cocktail, Sigma-Aldrich) and homogenized for 2 min by 30 Hz using Tissuelyser (Qiagen). The homogenates were centrifuged at 800 g for 15 min at 4 °C. The pellets were processed for nuclear extraction whereas the supernatants were used for subsequent isolation of cytosolic fraction. The pellet was resuspended in 200 μl of buffer B (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.5 M NaCl, 0.2 mM EDTA, 20% glycerol, 1% Triton-X-100, Protease Inhibitor Cocktail, Sigma-Aldrich) and incubated on ice for 30 min. The nuclei were lysated with 10–20 passages through an 18-gauge needle and finally the lysate was centrifuged at 9,000 g for 30 min and the nuclear fraction resulted in the supernatant. Both nuclear and cytosolic proteins, 20 μg per lane, were separated by SDS-PAGE.

Mannini L., Cucco F., Quarantotti V., Amato C., Tinti M., Tana L., Frattini A., Delia D., Krantz I.D., Jessberger R, & Musio A. (2015). SMC1B is present in mammalian somatic cells and interacts with mitotic cohesin proteins. Scientific Reports, 5, 18472.

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Protein Expression and Purification

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The plasmids of GST-fusion, MBP-tagged or 6× His-tagged proteins were transformed into E. coli strain BL21 or Rosetta (DE3), and expression was induced with 0.5 mM IPTG, with shaking overnight at 16°C. Bacteria expressing GST-SET7/9 and GST-PLK1 (WT, K191R, K474R/K492R) were suspended and lysed by sonication in PBS buffer containing protease inhibitor cocktail (Sigma; P8849) and 1 μg/ml PMSF, followed by purification using glutathione-Sepharose 4B (Sigma).
Bacteria expressing MBP-Bora were suspended and lysed by sonication in MBP column buffer (20 mM Tris–HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA) containing protease inhibitor cocktail (Sigma; P8849) plus 1 μg/ml PMSF, followed by use of amylose resin, as described previously (Seki et al., 2008 (link)).
Bacteria expressing 6×His-PLK1 were suspended and lysed by sonication in lysis buffer (50 mM NaH₂PO₄, pH 8.0, 150 mM NaCl, 10 mM imidazole) with protease inhibitor cocktail (Sigma; P8849) and 1 μg/ml PMSF. Then 6× His-tagged proteins were bound to Ni-NTA resin (Qiagen) and eluted with elution buffer (50 mM NaH₂PO₄, pH 8.0, 150 mM NaCl, 200 mM imidazole). All purified proteins were analyzed and confirmed by SDS–PAGE.

Yu R., Wu H., Ismail H., Du S., Cao J., Wang J., Ward T., Yang F., Gui P., Ali M., Chu L., Mo F., Wang Q., Chu Y., Zang J., Zhao Y., Ye M., Fang G., Chen P.R., Dou Z., Gao X., Wang W., Liu X, & Yao X. (2019). Methylation of PLK1 by SET7/9 ensures accurate kinetochore–microtubule dynamics. Journal of Molecular Cell Biology, 12(6), 462-476.

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Cell Fractionation and Lysis Protocol

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Cells were seeded in 6-well plates [1.0 × 10⁵ cells per well] and allowed to attach for 24 h. Cells were treated for 48 h with the indicated dose of inhibitor, then trypsinized and an equal number of cells collected from each sample for fractionation. Cells were resuspended in 300 μL of lysis buffer 1 (50 mM Hepes-KOH pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% TX-100) containing protease inhibitor cocktail (Sigma-Aldrich), and 100 μL of suspension was collected as whole lysate. Samples were centrifuged at 9,300 rcf for 10 sec and 100 μL supernatant collected as cytoplasmic lysate. Remaining cells were washed in lysis buffer 2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) containing protease inhibitor cocktail (Sigma-Aldrich), and resuspended in 100 μL lysis buffer 3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine) containing protease inhibitor cocktail (Sigma-Aldrich). All samples were sonicated at high intensity for 10 × 30 sec with a 1 min rest on ice followed by centrifugation at 15,700 rcf for 10 min.

Armstrong H.K., Gillis J.L., Johnson I.R., Nassar Z.D., Moldovan M., Levrier C., Sadowski M.C., Chin M.Y., Tomlinson Guns E.S., Tarulli G., Lynn D.J., Brooks D.A., Selth L.A., Centenera M.M, & Butler L.M. (2018). Dysregulated fibronectin trafficking by Hsp90 inhibition restricts prostate cancer cell invasion. Scientific Reports, 8, 2090.

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Protease inhibitor cocktail

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9 861 protocols using protease inhibitor cocktail

Co-Immunoprecipitation Protocols for Endogenous and Overexpressed Proteins

Co-Immunoprecipitation Protocols for Endogenous and Overexpressed Proteins

Fluorescence-Activated Nuclei Sorting of OPCs

Protein Extraction and Interaction Analysis

Immunoprecipitation and Immunoblotting of Nuclear Proteins

Cytosol and Nucleus Protein Fractionation

Protein Extraction and Interaction Analysis

Subcellular Protein Fractionation and Analysis

Protein Expression and Purification

Cell Fractionation and Lysis Protocol

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