To remove vesicular nucleic acids and proteins, 1 mg/mL of native LrEVs were treated with 10 U/mL of DNase I (Thermo Scientific, #EN0521), 10 U/mL of RNase I (Solarbio, #R8021), and 1 mg/mL of proteinase K-agarose (Sigma, #P9290), respectively, as described previously [16 (link), 31 (link)]. The native LrEVs were incubated with DNase I and RNase I at 37 °C for 30 min, and with proteinase K-agarose at 37 °C for 2 h. The enzymes were inactivated at 75 °C for 1 h. proteinase K-agarose was additionally removed by centrifugation (12,000×g, 1 min).
Proteinase k agarose
Manufactured by Merck Group
Proteinase K-agarose is a laboratory product used for the purification of nucleic acids. It is an enzyme immobilized on an agarose matrix, which is commonly used in the process of protein digestion during DNA or RNA extraction procedures.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Rnase 1, by Solarbio (1 mentions)
Centrifugal concentrator, by Merck Group (1 mentions)
Valinomycin, by Merck Group (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Lds loading buffer, by Thermo Fisher Scientific (1 mentions)
Lumiglo, by Cell Signaling Technology (1 mentions)
V5 hrp, by Thermo Fisher Scientific (1 mentions)
Histrap column, by GE Healthcare (1 mentions)
L arabinose, by Merck Group (1 mentions)
Superdex 200 10 300 gl column, by GE Healthcare (1 mentions)
E coli polar lipid extract, by Avanti Polar Lipids (1 mentions)
Egg phosphatidylcholine, by Avanti Polar Lipids (1 mentions)
Trypsin agarose, by Merck Group (1 mentions)
Gemini 5μ c18 110a column, by Phenomenex (1 mentions)
1100 series hplc, by Phenomenex (1 mentions)
Prism 8, by GraphPad (1 mentions)
5 protocols using proteinase k agarose
1
Vesicular Nucleic Acid and Protein Removal
2
Purification and Characterization of Membrane Proteins
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Isopropyl-β-d -thiogalactopyranoside (IPTG), Tris(2-carboxyethyl)phosphine (TCEP), n-dodecyl-β-d -maltopyranoside (DDM) and precast SDS–PAGE gels were from Generon. All aromatic acids, l -arabinose, cholesteryl hemisuccinate Tris salt (CHS), 0.4–0.6 mm acid-washed glass beads, valinomycin and Proteinase K-agarose were from Sigma–Aldrich. HisTrap columns, PD-10 and PD SpinTrap G-25 gel filtration columns, Superdex 200 10/300 GL column, size exclusion column standards, and nitrocellulose membrane were from GE Healthcare. Chemiluminescence reagents (LumiGLO) were from Cell Signaling Technologies. E. coli strain BL21-AI, LDS loading buffer, V5-HRP, pyranine and gels and reagents for blue native PAGE were from Life Technologies. Centrifugal concentrators were from Millipore. The detergent compatible Lowry assay was purchased in kit format from ThermoFisher. E. coli polar lipid extract and egg phosphatidylcholine were from Avanti Polar Lipids.
3
Optimizing Protein Binding via Proteolysis
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The cDNA fusions were incubated with proteinase K agarose, chymotrypsin agarose, and trypsin agarose (Sigma). Round 0-3 fusions were not subjected to proteases and round 4-6 were subjected to increasing amounts of each protease (0.1 mg proteinase K for round 4, 0.2 mg proteinase K for round 5, and 0.2 mg proteinase K, 0.2 mg chymotrypsin, 0.1 mg trypsin for round 6). All proteolysis was performed at RT. Proteases were removed by spin filtration.
4
Peptide Protease Stability Assay
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Protease stability was assessed by treating peptide solutions (1 mM in PBS, final volume 500 μL) with 0.3 mg proteinase-K agarose (Sigma). At time intervals of 0, 30, 60, 120, and 240 minutes, 50 μL samples were taken from the digestion mixture and separated from proteinase-K agarose by centrifugal filtration with Spin-X columns. Samples were then subjected to analytical HPLC (Agilent 1100 Series HPLC equipped with a Phenomenex Gemini 5μ C18 110A column) using gradient elution (10–30% buffer B over 30 minutes; buffer A dH2O + 0.1% (v/v) TFA, buffer B CH3CN + 0.1% (v/v) TFA). Peak area was calculated for the peak corresponding to the intact peptide for each sample and the fraction intact was calculated for each time point. Half-lives for each peptide were calculated in Graphpad Prism 8.
5
Saliva Protein Purification Protocol
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1. Heat-denaturation: Samples were boiled for 5 min and rapidly cooled on ice. 2. Proteinase-K treatment: Proteinase-K agarose (P9290, Sigma-Aldrich) was hydrated in 10mM sodium phosphate buffer at pH 7.0. Fifty μl of packed beads were added to 100 μl saliva proteins at 6.2 mg/ml and incubated for 4h at 37°C. The supernatant was diluted in DMEM to an equivalent of 1 mg/ml protein concentration for cell stimulation. 3. Jacalin capture: Jacalin agarose (Pierce/Thermo) was hydrated in PBS. Fifty μl of packed beads were added to 100 μl saliva proteins at 6.2 mg/ml and incubated for 4h at 4°C. The supernatant was diluted in DMEM to an equivalent of 1 mg/ml protein.
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