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Protease inhibitor cocktail and phosphatase inhibitor cocktail

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Protease inhibitor cocktail and phosphatase inhibitor cocktail are laboratory reagents used to inhibit the activity of proteases and phosphatases, respectively, in biological samples. These cocktails contain a mixture of compounds that target and inactivate a broad range of enzymes, preventing their degradation of proteins during sample preparation and analysis.

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Lab products found in correlation

Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions) Ripa lysis buffer, by Merck Group (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Protein a agarose, by Roche (1 mentions) Kdm4b, by Cell Signaling Technology (1 mentions) Eif2α, by Santa Cruz Biotechnology (1 mentions) P eif2α, by Cell Signaling Technology (1 mentions) P erk, by Cell Signaling Technology (1 mentions) Protein g agarose, by Roche (1 mentions) Lysosome isolation kit, by Merck Group (1 mentions) Ripa lysis buffer, by Beyotime (1 mentions) Bca protein assay kit, by Beyotime (1 mentions) Phospho rtk array, by R&D Systems (1 mentions) C myc 5605, by Santa Cruz Biotechnology (1 mentions) Anti mouse and anti rabbit hrp conjugated secondary antibodies, by Bio-Rad (1 mentions)

4 protocols using protease inhibitor cocktail and phosphatase inhibitor cocktail

1

Histone Isolation from Human Kidney

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Histone proteins were isolated from the human kidney tissues by acid extraction procedure [22 (link)]. Briefly, nuclei were obtained by homogenizing kidney tissues in hypotonic lysis buffer (10 mM Tris-Cl pH 8.0, 1 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol) with protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche), and histones were isolated from nuclei by extraction with 0.4 N H2SO4. For cell culture, cells were collected and lysed in radio immuno precipitation assay (RIPA) lysis buffer (Millipore) with protease inhibitor cocktail (Roche). The cell lysates were centrifuged, and the supernatants were collected and stored at −80 °C until analysis.
Li C., Choi H.P., Wang X., Wu F., Chen X., Lü X., Jing R., Ryu H., Wang X., Azadzoi K.M, & Yang J.H. (2017). Post-Translational Modification of Human Histone by Wide Tolerance of Acetylation. Cells, 6(4), 34.
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2

Coimmunoprecipitation Assay for Protein-Protein Interactions

Cited 1 time
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For immunoprecipitation, the following antibodies were used: KDM4B (Cell Signaling; #8639, 1:200 dilution), eIF2α (Santa Cruz; sc-133132, 1:200 dilution), PERK (Cell Signaling; #5683, 1:200 dilution), and P-eIF2α (Cell Signaling; #3398, 1:200 dilution). Coimmunoprecipitation assays were performed as described previously (Wee et al., 2015 (link)) with a modified protocol using NETN buffer (25 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 10% glycerol, and 0.3% NP-40) containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche Diagnostics) to extract whole-cell protein, or a cell fractionation protocol for cytoplasmic and nuclear fractionation. Cell lysates were incubated with the indicated antibodies at 4°C overnight. The protein complex was captured using protein A–agarose (for rabbit primary antibody) or protein G–agarose (for mouse primary antibody) beads (Roche Diagnostics) at 4°C for 4 h, and agarose beads were collected by centrifuge and washed three times with washing buffer. The precipitated proteins were dissolved in SDS sample buffer along with 3 mM dithiothreitol and subjected to Western blot analysis.
Wang W., Oguz G., Lee P.L., Bao Y., Wang P., Terp M.G., Ditzel H.J, & Yu Q. (2018). KDM4B-regulated unfolded protein response as a therapeutic vulnerability in PTEN-deficient breast cancer. The Journal of Experimental Medicine, 215(11), 2833-2849.
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3

Isolation and Characterization of Cellular Organelles

Cited 1 time
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Cell lysates were prepared from primary cultured microglia, neurons, HEK293T cells or brain tissue by RIPA lysis buffer (Beyotime, China) with protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche). Nuclear and cytoplasmic extracts were prepared by the NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, #78833) according to the manufacturer's instructions. Protein concentrations were determined using an enhanced bicinchoninic acid (BCA) protein assay kit (Beyotime, China) prior to Western blot or immunoprecipitation analysis. Lysosomes were isolated by a lysosome isolation kit (Sigma) with density gradient ultracentrifugation according to the manufacturer's description. Brain tissue was prepared from the ipsilateral parietal cortex (penumbra of the middle cerebral artery) or hippocampus and the corresponding area of the contralateral side 20 (link).
Zhou H., Yan L., Huang H., Li X., Xia Q., Zheng L., Shao B., Gao Q., Sun N, & Shi J. (2023). Tat-NTS peptide protects neurons against cerebral ischemia-reperfusion injury via ANXA1 SUMOylation in microglia. Theranostics, 13(15), 5561-5583.
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4

Western Blot Analysis of Signaling Proteins

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Cells were washed with ice-cold PBS and then lysed with RIPA lysis buffer (150 mmol/L Tris, pH 7.4, 100 mmol/L NaF, 120 mmol/L NaCl, 100 µmol/L sodium orthovanadate, and 1× protease inhibitor cocktail and phosphatase inhibitor cocktail; Roche). Lysates (30 µg) were resolved by SDS-PAGE and transferred to nitrocellulose membranes; which were incubated with primary antibodies at 4°C overnight or at room temperature for 2 hours followed by incubation with HRP-conjugated anti-rabbit and anti-mouse secondary antibodies (Bio-Rad) for 1 hour at room temperature. Immunoreactive bands were visualized by chemiluminescence (Santa Cruz Biotechnology). All primary antibodies were from Cell Signaling Technology with the exception of BRD4 (Abcam), Actin (Abcam) and AXL (Life Technologies). For phospho-RTK arrays (R&D Systems), lysates were procured and applied to arrays according to manufacturer’s instructions. Arrays were visualized using chemiluminescence. Antibodies used for immunoblotting were from Cell Signaling Technology (P-MET-Y1234 #3126, P-CDK9 #2549, P21 #2947, HER3 #12708, P-HER3-Y1289 #4791, ROR2 #88639, HER2 #2242, P-SFK-Y419 #6943, C-MYC #5605, P-MAPK #9101, GAPDH #5174), Santa Cruz Biotechnology (V5 probe #sc-271944), R&D Systems (P-AXL-Y779 #AF2228, AXL #AF154), Abcam (BRD4 #128874, c-MET #59884, Actin #6276, Beta-tubulin #6046) and BD Biosciences (EGFR #610016).
Leonard B., Brand T.M., O’Keefe R.A., Lee E.D., Zeng Y., Kemmer J.D., Li H., Grandis J.R, & Bhola N.E. (2018). BET inhibition overcomes receptor tyrosine kinase-mediated cetuximab resistance in HNSCC. Cancer research, 78(15), 4331-4343.
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