Proteinase k

Manufactured by Worthington

Sourced in United States

Proteinase K is a serine protease enzyme that is commonly used in molecular biology laboratories. It functions to hydrolyze and degrade a wide range of proteins, including those found in cell membranes and nuclei. Proteinase K is known for its ability to effectively and efficiently break down proteins, making it a valuable tool in various biological applications.

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Lab products found in correlation

Picogreen, by Thermo Fisher Scientific (2 mentions) Facscanto analyzer, by BD (2 mentions) Sytox green, by Thermo Fisher Scientific (2 mentions) Proteinase k, by Merck Group (2 mentions) Biotinylated anti mouse igg antibody, by Vector Laboratories (2 mentions) Collagenase 1, by Worthington (1 mentions) E210 device, by Covaris (1 mentions) Ab6672, by Abcam (1 mentions) Af7014, by Affinity Biosciences (1 mentions) 3 3 diaminobenzidine solution, by Nichirei Biosciences (1 mentions) Bloxall blocking solution, by Vector Laboratories (1 mentions) Abc kit, by Vector Laboratories (1 mentions) Vectastain elite abc rabbit igg kit, by Vector Laboratories (1 mentions) Dako liquid dab substrate chromogen system, by Agilent Technologies (1 mentions) Bz x710, by Keyence (1 mentions) Dreamtaq green pcr master mix, by Thermo Fisher Scientific (1 mentions) 4 2 aminoethyl benzenesulfonyl fluoride hydrochloride, by Merck Group (1 mentions) Trypsin, by Merck Group (1 mentions) Trypsin, by Promega (1 mentions) Confocal fluorescence microscope, by Zeiss (1 mentions)

16 protocols using proteinase k

Cell Proliferation Quantification in Corneal and Stromal Scaffolds

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For cell proliferation, the DNA of cLESCs seeded in SF/G corneal film (100,000 cell/cm²) and cCSSCs seeded in SF/G stromal scaffold (cell concentration 6x10⁶ cells/mL; 80 μl/scaffold) were quantified at days 1, 3, 5, 7, 14, and 28. The samples of each time point were digested with 1.0 mg/mL proteinase K (Worthington Biochemical, USA) in proteinase K buffer (150 mM Tris HCl and 1 mM EDTA pH 8.0) and 20 μl/sample of papain suspension (Worthington Biochemical, USA). After that, all samples were incubated at 60°C for 16 h in a heating block. Centrifugation was performed with 8,000 rpm for 10 min at 4°C to get the supernatant and transferred to a new tube. DNA levels were measured by Qubit Fluorometric Quantification with dsDNA BR assay according to the manufacture’s protocols. Quantitative data was normalized with plain film and scaffold.

Torsahakul C., Israsena N., Khramchantuk S., Ratanavaraporn J., Dhitavat S., Rodprasert W., Nantavisai S, & Sawangmake C. (2022). Bio-fabrication of stem-cell-incorporated corneal epithelial and stromal equivalents from silk fibroin and gelatin-based biomaterial for canine corneal regeneration. PLoS ONE, 17(2), e0263141.

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Chromatin Immunoprecipitation (ChIP) Protocol

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ChIP was performed as described.^{59 (link)} Briefly, nuclei were isolated from the x-linked cells and were sonicated using Bioruptor (Diagnode), according to the manufacturer’s protocol. A total of 8mg of sheared crosslinked chromatin was incubated with 8μg of antibody pre-loaded on a 1:1 ratio of protein A and protein G magnetic beads (Life Technologies, 10002D and 10004D, respectively). After washing the beads, the samples were eluted in 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0. Crosslinking was reversed by incubation at 65°C for 30 min with shaking. Samples were treated with RNase (Roche, 11119915001) for 2 h at 37°C and then treated with Proteinase K (Worthington, LS004222) for 2 h at 55°C. DNA was purified by phenol: chloroform extraction followed by ethanol precipitation and quantified using PicoGreen (Life Technologies, P11495) and NanoDrop 3300 fluoro spectrometer. qPCR and data analysis were performed as previously described.^{59 (link)} Enrichment was calculated as follows = Ct (IN)-Ct (IP) and the fold enrichment over input = 2^[Ct (IN)-Ct (IP)]. Fold change was calculated by normalizing the fold enrichment at a specific site to that at the control region (Chr 17: 13821873–13821988). The significance of the change was determined via p value, which was calculated by GraphPad Prism using Student’s t-test. Table S4 lists sequences of primers used.

Miller W.G., Bates A.H., Horn S.T., Brandl M.T., Wachtel M.R, & Mandrell R.E. (2000). Detection on Surfaces and in Caco-2 Cells of Campylobacter jejuni Cells Transformed with New gfp, yfp, and cfp Marker Plasmids. Applied and Environmental Microbiology, 66(12), 5426-5436.

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Enzymatic Degradation of Biomaterials

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The ability of biomaterials to undergo enzymatic degradation was assessed in accordance with a procedure described previously [34 (link)]. Nevertheless, for this experiment, solutions of collagenase I (320 μg/mL) or proteinase K (0.125 μg/mL) were used (both enzymes were supplied by Worthington Biochemical Corporation, Lakewood, NJ, USA). Subsequently, the biomaterials were immersed in 5 mL of enzyme solutions or PBS (control experiment), and incubated for 3, 6, and 9 weeks at 37 °C with constant agitation—50 rpm (New Brunswick^TM Innova^® 42 Incubator Shaker, Eppendorf, Warsaw, Poland). Every three weeks, the solutions of enzymes or PBS were replaced with new portions. After incubation time, the biomaterials were rinsed with PBS, air-dried, and weighed. The ability of biomaterials to degrade was assessed by the measurement of differences in the biomaterial’s weight before and after incubation in the tested solutions as described previously [34 (link)].

Klimek K., Palka K., Truszkiewicz W., Douglas T.E., Nurzynska A, & Ginalska G. (2022). Could Curdlan/Whey Protein Isolate/Hydroxyapatite Biomaterials Be Considered as Promising Bone Scaffolds?—Fabrication, Characterization, and Evaluation of Cytocompatibility towards Osteoblast Cells In Vitro. Cells, 11(20), 3251.

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ChIP-qPCR Quantification Protocol

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ChIP was performed as described (Petell et al., 2016 (link)). Chromatin was sheared by sonication using a Covaris E210 device, according to the manufacturer’s protocol. A total of 8mg of sheared crosslinked chromatin was incubated with 8mg of antibody pre-loaded on a 1:1 ratio of protein A and protein G magnetic beads (Life Technologies, 10002D and 10004D, respectively). After washing the beads, the samples were eluted in 1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0. Crosslinking was reversed by incubation at 65°C for 30 min with shaking. Samples were treated with RNase (Roche, 11119915001) for 2 h at 37°C, and subsequently treated with Proteinase K (Worthington, LS004222) for 2 h at 55^°C. DNA was purified by phenol:chloroform extraction followed by ethanol precipitation and quantified using PicoGreen (Life Technologies, P11495) and NanoDrop 3300 fluorospectrometer. qPCR was then performed using equal amounts of IN and IP samples. Fold enrichment was first calculated as:

2^{\land} (C_{t} (IN) - C_{t} (IP)) .

Percent enrichment = Fold enrichment X100. Significance of change was determined via p-value, which was calculated by GraphPad Prism using Student’s t test.
Table S4 lists sequences of primers used.

AlAbdi L., Saha D., He M., Dar M.S., Utturkar S.M., Sudyanti P.A., McCune S., Spears B.H., Breedlove J.A., Lanman N.A, & Gowher H. (2020). Oct4-Mediated Inhibition of Lsd1 Activity Promotes the Active and Primed State of Pluripotency Enhancers. Cell reports, 30(5), 1478-1490.e6.

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Immunohistochemical Analysis of Inflammatory Markers

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Deparaffinization sections were washed thrice for 5 min each with phosphate buffered saline (PBS) (pH 7.4). For antigen activation, Proteinase K (Worthington Biochemical Co., Lakewood, NJ, USA)/distilled water (0.2 mg/mL) was added dropwise to the sections, which were then incubated for 15 min. After rewashing with PBS, endogenous peroxidase activity was blocked by BLOXALL blocking solution (Vector Laboratories, Newark, CA, USA) for 10 min. Blocking used 0.05% normal goat serum/PBS solution for 1 h. After rewashing again with PBS, the following primary alternatives were reacted at 4 °C overnight: antitumor necrosis factor-alpha (TNF-α), rabbit polyclonal antibody (1:200 dilution, AF7014; Affinity Biosciences, Solon, OH, USA), and anti-interleukin-6 (IL-6) rabbit polyclonal antibody (1:100 dilution, ab6672; Abcam, Cambridge, MA, USA). Subsequently, the streptavidin-biotin-peroxidase complex technique was performed using an ABC kit (Vector Laboratories). After rewashing with PBS, the sections were colored brown using a 3,3-diaminobenzidine solution (NICHIREI Biosciences, Tokyo, Japan). Nuclei were counterstained with Mayer’s hematoxylin. Immunohistochemically (IHC) stained images were calculated as the ratio of positive cells per unit area using the Fiji image analysis software [18 (link)].

Ozone K., Minegishi Y., Oka Y., Sato M, & Kanemura N. (2023). The Effects of Downhill Running and Maturation on Histological and Morphological Properties of Tendon and Enthesis in Mice. Biology, 12(3), 456.

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Yeast Cell Permeabilization and DNA Staining

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Fixed yeast cells were washed with water, briefly sonicated, and incubated in 50 mM sodium citrate (pH 7.4) with 0.3 mg/mL RNase A for 2 h at 50°C. Then, 0.6 mg/mL Proteinase K (Worthington) was added and incubated for an additional 2 h at 50°C. Finally, cell pellets were resuspended in 50 mM sodium citrate with 1:5,000 SYTOX green (Invitrogen) and incubated for 1 h at room temperature. Flow cytometry was performed on a BD FACSCanto analyzer, and 30,000 cells were recorded for each sample.

Li Y., Hartemink A.J., & MacAlpine D.M. (2021). Cell cycle-dependent chromatin dynamics at replication origins.

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Immunohistochemical Analysis of TNF-α and MMP-13 in Articular Cartilage

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To evaluate the expression of TNF-α and MMP-13, we performed immunohistochemical staining following the avidin-biotin complex method using the VECTASTAIN Elite ABC Rabbit IgG Kit (Vector Laboratories, Burlingame, CA). The tissue sections were deparaffinized with xylene and ethanol, and antigen activation was performed using proteinase K (Worthington Biochemical Co., Lakewood, NJ) for 15 min. Endogenous peroxidase was inactivated with 0.3% H₂O₂/methanol for 30 min. Nonspecific binding of the primary antibody was blocked using normal goat serum for 30 min, and then the sections were incubated with anti-TNF-α and anti-MMP-13 primary antibodies overnight at 4°C. Afterward, the sections were incubated with biotinylated secondary anti-rabbit IgG antibody and stained using Dako Liquid DAB+ Substrate Chromogen System (Dako, Glostrup, Denmark). Cell nuclei were stained with hematoxylin. For analysis, all the samples were taken with fluorescence microscope BZ-X710(Keyence, Tokyo, Japan), and the articular cartilage was taken at 40x magnification. We calculated the ratio of the number of TNF-α- or MMP-13-positive cells to the number of chondrocytes in the articular cartilage area of 10,000 μm^{2 (link)} (100 μm × 100 μm) on 1 slide of each sample.

Arakawa K., Takahata K., Enomoto S., Oka Y., Ozone K., Morosawa K., Murata K., Kanemura N, & Kokubun T. (2022). Effect of Suppression of Rotational Joint Instability on Cartilage and Meniscus Degeneration in Mouse Osteoarthritis Model. Cartilage, 13(1), 19476035211069239.

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Genomic DNA Extraction and PCR Amplification

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Genomic DNA was isolated from the tail tip of each mouse, and processed using 100 μl lysis buffer (50 mM KCl, 10 mM Tris, 2.5 mM MgCl₂, 0.45% NP-40, 0.45% Tween-20) freshly supplemented with 0.1 mg/ml Proteinase K (LS004222; Worthington Biochemical Corporation, Lakewood, JN, US). DNA samples were then subjected to PCR amplification in a 20 μl reaction mixture consisting of 10 μl DreamTaq Green PCR Master Mix (K1082; Thermo Scientific, Waltham, MA, US), 0.5 μl forward primer, 0.5 μl reverse primer, 2 μl sample DNA, and 7 μl DNAase-free water. The reaction was initiated at 95 ℃ for 4 minutes, followed by 35 cycles of 95 ℃ for 30 seconds, 59 ℃ for 30 seconds and 72 ℃ for 35 seconds, completed at 72 ℃ for 5 minutes. The PCR products were examined in 2.5% agarose gel stained with ethidium bromide (80 V, 30 minutes), and visualized by UV light. PCR primers are 5’-ATGCACGGCCACTACCACTAAGGCTT-3’ (forward) and 5’-AGATTTAGTGGCCAAGTACCTGCCAC-3’ (reverse).

Li Y., Shi Z., Jules J., Chen S., Kesterson R.A., Zhao D., Zhang P, & Feng X. (2019). Specific RANK Cytoplasmic Motifs Drive Osteoclastogenesis. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 34(10), 1938-1951.

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Crosslinked Protein Digestion Protocols

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Crosslinked proteins were digested with either trypsin (Promega) for 18 h at 37 °C or proteinase K (Worthington Biochemical) for 120 min at 37 °C. Both proteases were used at a 1:20 protease to protein ratio. trypsin and proteinase K digestions were inhibited by the addition of 10 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (Sigma-Aldrich).

Groitl B., Horowitz S., Makepeace K.A., Petrotchenko E.V., Borchers C.H., Reichmann D., Bardwell J.C, & Jakob U. (2016). Protein unfolding as a switch from self-recognition to high-affinity client binding. Nature Communications, 7, 10357.

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In Situ DNA Fragmentation Detection

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For in situ detection of DNA fragmentation in paraffin-embedded tissue sections, the TUNEL method was performed using the In Situ Cell Death Detection Kit, POD (Roche, Basel, Switzerland). Briefly, tumor sections were adhered to silane-coated slides and allowed to dry at 60 °C oven overnight. Subsequently, slides were deparaffinized and rehydrated. Protein digestion was done by incubating tissue in 20 μg/ml proteinase K (Worthington biochemical Co., Lakewood, NJ) for 15 min at room temperature. Antigen retrieval was done by immersing slides in 0.01 M citrate buffer, pH 6.0, in a microwave 750W for 1 min, followed by rapid cooling (30 min). Subsequently, the slides were immersed in Tris-HCl, 0.1 M pH 7.5, containing 3% BSA and 20% normal bovine serum at room temperature for 30 min. TUNEL reaction mixture was added to slides and incubated at 37 °C in a humidified chamber for 1 hr. Tumor cell nuclei were stained with DAPI 32670 (Sigma Co., Steinheim, Germany). After extensive washing, the slides were mounted onto glass coverslips, and analyzed using an excitation wavelength in the range of 450–500 nm and detection in the range of 515–565 nm (green) by fluorescence confocal microscope (Zeiss, Jena, Germany).

Chen N.C., Chyau C.C., Lee Y.J., Tseng H.C, & Chou F.P. (2016). Promotion of mitotic catastrophe via activation of PTEN by paclitaxel with supplement of mulberry water extract in bladder cancer cells. Scientific Reports, 6, 20417.

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