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Protease inhibitor cocktail mix

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Protease inhibitor cocktail mix is a laboratory reagent that contains a combination of small-molecule compounds that inhibit the activity of various proteases. This mixture is commonly used in protein extraction and purification procedures to prevent the degradation of target proteins by endogenous proteases present in biological samples.

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Lab products found in correlation

Ripa buffer, by Merck Group (3 mentions) Pierce bicinchoninic acid bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions) Phenylpyruvic acid, by Merck Group (1 mentions) 2 amino 2 methyl 1 3 propanediol, by Merck Group (1 mentions) Pyridoxal 5 phosphate hydrate, by Merck Group (1 mentions) Phenylmethanesulfonyl fluoride, by Merck Group (1 mentions) Se methyl selenocysteine hydrochloride, by Merck Group (1 mentions) 2 4 dinitrophenylhydrazine, by Merck Group (1 mentions) L phenylalanine, by Merck Group (1 mentions) Nadph, by Thermo Fisher Scientific (1 mentions) Indole 3 pyruvic acid, by Merck Group (1 mentions) Emem media, by American Type Culture Collection (1 mentions) Hepg2 cell, by American Type Culture Collection (1 mentions) Plko 1 vector, by Merck Group (1 mentions) Pegfp n1, by Takara Bio (1 mentions) N n dimethylformamide (dmf), by Merck Group (1 mentions) Dimethyl 2 oxoglutarate, by Merck Group (1 mentions) Bff 122, by Axon Medchem (1 mentions) Precellys 24, by Bertin Technologies (1 mentions)

6 protocols using protease inhibitor cocktail mix

1

Protein Extraction from Cultured Cells

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Samples were lysed in ice for 30 min in RIPA buffer (Sigma, Darmstadt, Germany) in the presence of 1 mM PMSF and 1% Protease inhibitor cocktail mix (Sigma). Further, lysed cells were sonicated at 4 °C for 30 s with 1- to 2-s pulses. The proteins were harvested by centrifuging at 13,000 rpm for 10 min at 4 °C and the supernatant was collected, and protein concentration was determined by the Pierce™ Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fischer Scientific, Rockford, IL, USA) according to the manufacturer’s protocols.
Selvam A.K, & Björnstedt M. (2020). A Novel Assay Method to Determine the β-Elimination of Se-Methylselenocysteine to Monomethylselenol by Kynurenine Aminotransferase 1. Antioxidants, 9(2), 139.
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2

CaMKII Phosphorylation Analysis

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Proteins were prepared with Pierce™ RIPA Buffer (Thermoscientific catalog # 89900) according to the manufacturer’s protocol, with a protease inhibitor cocktail mix (Sigma catalog # P8340). Protein concentrations were verified using the BCA assay. Western Blotting was essentially carried out as previously described, with modifications, as described in Additional file 1: Expanded Methods. Phosphorylation of CaMKII (CaMK2) was analyzed by comparing the phosphorylated and unphosphorylated proteins, which are also described in more detail in the expanded methods section.
Aguilan J.T., Pedrosa E., Dolstra H., Baykara R.N., Barnes J., Zhang J., Sidoli S, & Lachman H.M. (2023). Proteomics and phosphoproteomics profiling in glutamatergic neurons and microglia in an iPSC model of Jansen de Vries Syndrome. bioRxiv.
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3

Cell Lysis and Protein Extraction

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Samples were lysed in RIPA buffer (Sigma, Darmstadt, Germany) in the presence of 1 mM phenylmethanesulfonyl fluoride (PMSF) and 1% protease inhibitor cocktail mix (Sigma, Darmstadt, Germany) or 100 mM potassium phosphate EDTA buffer pH 8.0 (for intracellular thiol determination). Further, lysed cells were sonicated at 4 °C for 30 s with 1- to 2-s pulses. The proteins were harvested by centrifugation at 13,000 rpm for 10 min at 4 °C, the supernatant was collected, and protein concentration was determined by the Pierce™ Bicinchoninic acid (BCA) Protein Assay Kit (Thermo Fischer Scientific, Rockford, IL, USA) according to the manufacturer’s instructions.
Selvam A.K., Jawad R., Gramignoli R., Achour A., Salter H, & Björnstedt M. (2021). A Novel mRNA-Mediated and MicroRNA-Guided Approach to Specifically Eradicate Drug-Resistant Hepatocellular Carcinoma Cell Lines by Se-Methylselenocysteine. Antioxidants, 10(7), 1094.
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4

Potassium Phosphate Metabolic Assay

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Potassium dihydrogen phosphate, di-potassium hydrogen phosphate, EDTA, sodium hydroxide, aminooxyacetic acid (AOAA), l-tryptophan (l-Try), indole-3-pyruvic acid (IPA), phenylpyruvic acid (PPA), α-Keto-γ-methylthiobutyric acid sodium salt (KMB), se-methylselenocysteine hydrochloride (MSC), dimethyl-2-oxoglutarate (α-KG), l-phenyl alanine (l-Phe), 2-amino-2-methyl-1,3-propanediol, pyridoxal 5′-phosphate hydrate (PLP), 2,4-dinitrophenylhydrazine (DNPH), Phenylmethanesulfonyl fluoride (PMSF), RIPA buffer, protease inhibitor cocktail mix, and N-N-dimethyl formamide, pLKO.1 vector were purchased from Sigma-Aldrich (Darmstadt, Germany). BFF-122 was purchased from Axon Medchem (Groningen, Netherlands), NADPH was purchased from Acros Organics (New Jersey, NJ, USA). Plasmid pEGFP-N1 (Clontech, Takara Bio USA, Inc, Mountain View, CA, USA) was a generous gift from Dr. Gildert Lauter from the Department for Biosciences and Nutrition, Karolinska Institutet, Stockholm, Sweden. HEPG2 cells and EMEM media were purchased from ATCC (Wesel, Germany).
Selvam A.K, & Björnstedt M. (2020). A Novel Assay Method to Determine the β-Elimination of Se-Methylselenocysteine to Monomethylselenol by Kynurenine Aminotransferase 1. Antioxidants, 9(2), 139.
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5

Preparation of Cell Homogenates from Infected Cells

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To prepare cell homogenates from chronically infected and uninfected clones, the cells were grown in 15 cm petri dishes to 80–90% confluency. Cells were resuspended in serum-free medium and then centrifuged for 5 min. at 500× g. Cell pellets were then resuspended in 1 mL of serum-free medium, supplemented with protease inhibitor cocktail mix (Merck Millipore, Watford, UK), 4 μL benzonase (25–29 U/µL, Merck Millipore), and zirconium beads. The cells were homogenized with two 60-s cycles at 6500 rpm while using a ribolyzer (Precellys 24, Bertin Instruments, Basingstoke, UK). The homogenates were stored at −80 °C until further processing.
Philiastides A., Ribes J.M., Yip D.C., Schmidt C., Benilova I, & Klöhn P.C. (2019). A New Cell Model for Investigating Prion Strain Selection and Adaptation. Viruses, 11(10), 888.
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6

Western Blot Analysis of Cell Lysates

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Western blot analyses were performed as described (Grindel et al., 2014 (link)). After culture for 24 hr on various substrates, cell lysate was collected with RIPA buffer containing 150 mM NaCl, 1.0% (v/v) Triton-X-100, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) sodium dodecyl sulfate (SDS), 10% (v/v) glycerol, 1mM ethylenediaminetetraacetic acid (EDTA), 1mM ethylene glycol tetraacetic acid (EGTA), 50 mM Tris, pH 8.0, protease inhibitor cocktail mix (EMD Millipore) at 1:100 dilution and phosphatase inhibitor cocktail (EMD Millipore) mix at 1:100 dilution. Protein extract was denatured at 99°°C with Pierce™ lane marker reducing sample buffer (Thermo Fisher Scientific) supplemented with 2% (v/v) 2-mercaptoethanol, then separated by SDS-polyacrylamide gel electrophoresis (PAGE) as previously described (Grindel et al., 2014 (link)). GADPH was used at concentration of 1:5,000 and all other antibodies were used at 1:1,000. The secondary antibodies goat anti-rabbit or sheep anti-mouse HRP conjugated antibody (Jackson) were used at 1:50,000. Blots were exposed to X-ray film, and signal intensities were quantitated using ImageJ software. Pixel optical densitometric values were obtained for each protein band signal and normalized to respective loading control.
Martinez J.R., Grindel B.J., Hubka K.M., Dodge G.R, & Farach-Carson M.C. (2018). Perlecan/HSPG2: Signaling Role of Domain IV in Chondrocyte Clustering with Implications for Schwartz-Jampel Syndrome. Journal of cellular biochemistry, 120(2), 2138-2150.
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