Previously isolated bacterial isolates from various fermented foods of North East India was evaluated for bacteriocin production by agar well diffusion method (Li et al., 2015 (link)). Cells were grown in MRS for 16–18 h and then centrifuged at 8000 rpm, 4°C for 15 min to remove the cells. The supernatant was neutralized to the pH of 7.0 with 0.1 N NaOH followed by filter-sterilization through 0.2 μm membrane. Consequently agar well diffusion assay was performed against indicator strain K. rhizophila. Briefly, 50 μL of cell free supernatants were placed into 6 mm wells on BHI agar plates seeded with the above indicator strains. After incubation at 37°C for 12 h, the diameters of inhibition zones were measured. Proteinaceous nature of antibacterial substance was checked by incubating the cell-free supernatant (CFS) with 1 mg/ml of proteinase K (Hi Media) at 37°C for 2 h (Devi and Halami, 2011 (link)). Both protease-treated CFS were assayed for activity as indicated above (Devi and Halami, 2011 (link)). CFS was also tested for heat resistance at boiling temperature for 15 min (Sure et al., 2016 ).
Proteinase k
Manufactured by HiMedia
Sourced in India
Proteinase K is a broad-spectrum serine protease enzyme commonly used in molecular biology and biochemistry laboratories. It functions by cleaving peptide bonds in a wide range of proteins, including those found in cell membranes and nucleic acid-protein complexes. Its primary purpose is to facilitate the isolation and purification of nucleic acids, such as DNA and RNA, from various biological samples.
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Lab products found in correlation
Foetal bovine serum (fbs), by HiMedia (1 mentions)
Agarose, by Merck Group (1 mentions)
Cell culture dishes and plates, by Corning (1 mentions)
Phosphate buffer saline (pbs), by HiMedia (1 mentions)
Ethidium bromide, by Merck Group (1 mentions)
Acridine orange, by Merck Group (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Merck Group (1 mentions)
Caspase 9 colorimetric kit, by Thermo Fisher Scientific (1 mentions)
Jc 1 dye, by BD (1 mentions)
Mitotracker red, by BD (1 mentions)
Trypan blue, by HiMedia (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
Sodium azide, by HiMedia (1 mentions)
Trypsin, by HiMedia (1 mentions)
Dimethyl sulfoxide (dmso), by HiMedia (1 mentions)
Rnase a, by HiMedia (1 mentions)
Phosphate buffered saline (pbs), by HiMedia (1 mentions)
Hydroxyproline, by HiMedia (1 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (1 mentions)
Quercetin, by Merck Group (1 mentions)
6 protocols using proteinase k
1
Bacteriocin Screening of North East Indian Fermented Foods
2
Assessment of Manikya Bhasma's Anticancer Potential
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Manikya Bhasma was obtained from local Baidyanath store in Guwahati city, acridine orange, propidium iodide, ethidium bromide, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), agarose, 2,7-dichlorofluorescein diacetate (DCFH-DA), dulbecco’s modified eagle’s medium were purchased from Sigma Aldrich (St. Louis, MO, USA). RNase A, Proteinase K, DMSO, Foetal Bovine Serum (FBS), Penicillin-Streptomycin (100X) antibiotic solution, Phosphate Buffer Saline (PBS), sodium azide, trypan blue, and trypsin were obtained from HiMedia (Mumbai, India). Ethylenediaminetetraacetic acid (EDTA), ethanol, sodium chloride, was purchased from Merck, Germany. Anti-Cyt-c antibodies, Mitotracker Red, and JC-1 dye were obtained from BD-Biosciences (San Jose, USA). The caspase-9 colorimetric kit was obtained from Invitrogen Corporation (Waltham, USA). All the cell culture plates and dishes were purchased from Corning, Lowell, MA, USA. MDAMB-231, DLD-1, HCT-116, MG-63, HeLa cancer cell lines were procured from National Centre for Cell Sciences, Pune, India. All other reagents and chemicals were of analytical grade purity.
3
Antioxidant Assay Protocol
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Phosphate-buffered saline, hydroxyproline, Ehrlich’s reagent, and proteinase K were purchased from the HiMedia Laboratories (Mumbai, India). Silver nitrate, quercetin, and DPPH were purchased from Sigma-Aldrich (St. Louis, MI, USA).
4
Thermostability and Protease Sensitivity of Antifungal Metabolites from Bacillus siamensis
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The thermostability of the antifungal metabolites produced by the endophyte Bacillus siamensis CNE6 was studied by keeping the CFS in a boiling water bath for 10 min. In addition, to check the proteinaceous nature of the antifungal principles, CFS was treated with proteinase K (1 mg/mL) (HiMedia, India) for 2 h at 37°C (48 (link)). The antifungal potential of both heat-killed and proteinase K-treated CFS was studied against the isolated pathogen CRP1 by using the agar well diffusion method (44 (link)). Untreated CFS was used as the control.
5
FFPE RNA Extraction and qPCR Analysis
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From FFPE (Formalin-fixed, paraffin-embedded) blocks, 5–8 curls were deparaffinized in xylene at 50°C, followed by proteinase K (HiMedia, India) treatment prior to RNA isolation. Either from lymphocytes or from deparaffinized retrospective samples [37 (link)] RNA was isolated by TRIzol™ (Ambion, US) method and quantified with Qubit RNA BR Assay Kit (Thermo Fisher Scientific, US) before converting to cDNA using SuperScript IV (SSIV, Thermo Fisher Scientific, US) as per manufacturers’ instructions. With no-template control (NTC), qPCR was performed in triplicates for each sample using KAPA SyBr green Universal reagents (Sigma Aldrich, US), cDNA (1:10 dilution) and primers in a 5μL reaction mix (qPCR condition: pre-incubation at 95°C for 10 minutes followed by amplification for 40 cycles–denaturation at 95°C for 10 sec, amplification at 60°C for 15 sec, and extension at 72°C for 15 sec) in Roche LightCycler 480 II machine.
6
Cell Cytotoxicity Assay Protocol
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Tris-HCl, NaCl, EDTA, SDS, Triton X-100, dimethyl sulfoxide (DMSO), formaldehyde and, DNase free-RNase-A were purchased from Merck Co. Mumbai, India. Propidium iodide (PI) was obtained from Beckton Dickinson, Gurgaon, Haryana, India. Dulbecco's Modified Eagles Medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin and trypsin-EDTA were purchased from Gibco BRL (Life technologies, Grand Island, NY, USA). 3-(4, 5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) reagent was obtained from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Crystal violet and proteinase K were obtained from Hi-Media, Mumbai, India. Hind III digest λ DNA (catalogue number D-4521) was obtained from Sigma -Aldrich, St. Louis, MO, USA.
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