The proteinase K digestion protection assay was per- formed as described in [74 (link)] with some modifications. Briefly, wild-type or the E530A variant of FK- JcR2a RNA was electroporated into Huh7.5 cells. The cells were collected at 48 h pe in PBS, subsequently resuspended in proteinase K buffer (50mM Tris-Cl, pH 8.0, 10 mM CaCl2, 1 mM DTT) and subjected to 5 freeze-thaw cycles. Cell lysates were obtained after centrifugation, divided into three groups: untreated or treated with 50 μg/μl of proteinase K (Roche, Mannheim, Germany) on ice for 1 h, and pretreated with 5% (vol/vol) Triton X-100 before proteinase K treatment on ice for 1 h. proteinase K was then inactivated with 5 mM phenylmethylsulfonyl fluoride (PMSF) on ice for 10 min and cell lysates were mixed with sample buffer. The amount of core protein was determined by Western blot.
Proteinase k
Manufactured by Roche
Sourced in Germany, Switzerland, United States, France, United Kingdom, Australia, Spain, Japan, China
Proteinase K is a serine protease that is commonly used in molecular biology and biochemistry laboratories. It is an enzyme that can effectively break down and digest proteins, making it useful for various applications such as DNA and RNA extraction, protein purification, and sample preparation for analysis.
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Lab products found in correlation
Rnase a, by Roche (22 mentions)
In situ cell death detection kit, by Roche (13 mentions)
Rnase a, by Merck Group (12 mentions)
Trizol, by Thermo Fisher Scientific (11 mentions)
Directpcr lysis reagent, by Viagen Biotech (11 mentions)
Phenol chloroform isoamyl alcohol, by Merck Group (11 mentions)
Nbt bcip, by Roche (11 mentions)
Rnase a, by Thermo Fisher Scientific (11 mentions)
Proteinase k, by Merck Group (10 mentions)
Whatman, by Cytiva (10 mentions)
Rnase a, by Qiagen (9 mentions)
Bm purple, by Roche (8 mentions)
Dnase 1, by Roche (8 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (7 mentions)
Dneasy blood and tissue kit, by Qiagen (7 mentions)
Mnase, by Takara Bio (7 mentions)
Te buffer, by Merck Group (7 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (7 mentions)
Bioruptor, by Diagenode (7 mentions)
Rnase free dnase, by Roche (6 mentions)
826 protocols using proteinase k
1
Proteinase K Digestion Assay for FKJcR2a
2
SAFB1 Knockout in HAP1 Cells
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PX330 or PX330-B/B was used for genome editing of the HAP1 cells. To establish SAFB1 knockout HAP1 cells, the sgRNA (5'-CGAAATTGAAATTACCTCCG-3') was selected from GeCKO v2 libraries [14] and cloned into the BbsI site of PX330 (Addgene). The plasmids (2 µg) and pcDNA6/TR plasmids (0.2 µg) were transfected similarly using a Nucleofector device (Lonza). To enrich the plasmid-transfected cells, the HAP1 cells were treated with 20 µg/ml blasticidin (InvivoGen) for 3 days, starting 1 day after transfection. Subsequently, the cells were diluted into 96-well plates for selection of single clones. The selected clones were lysed by proteinase K treatment (200 µg/ml proteinase K [Roche], 20 mM Tris-HCl pH 8.0, 5 mM EDTA, 400 mM NaCl, and 0.3% SDS) at 55°C for 1 hour, followed by proteinase K inactivation at 95°C for 15 min. To detect deletions or insertions, the lysates were subjected to PCR analysis using KOD FX Neo enzyme (TOYOBO) to amplify the genomic regions flanking the guide RNA target sites. To detect small deletions in the SLTM knockout cell lines, a T7 endonuclease I (NEB)
cleavage assay was performed on the amplified PCR products. The indel-positive clones were further confirmed by sequencing. Finally, the absence of protein expression was confirmed by immunoblotting.
cleavage assay was performed on the amplified PCR products. The indel-positive clones were further confirmed by sequencing. Finally, the absence of protein expression was confirmed by immunoblotting.
3
In Situ Hybridization and Immunohistochemistry
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In situ hybridization was carried out as previously described using an anti-sense probe against Ets-related gene (Erg) [41 (link),48 (link)]. For experiments in which in situ hybridization was coupled with immunohistochemistry, changes to the published in situ protocol were made as follows: 1) Embryos were fixed in 4% paraformaldehyde (PFA) for two hours at room temperature or 4° overnight then stored in 1X phosphate buffered solution (PBS); 2) Fixed embryos were rinsed three instead of five times in PBS-T (0.1% Tween-20) prior to Proteinase K treatment; 3) Embryos were incubated in 10 μg/mL Proteinase K (Roche) for 10 minutes; 4) Embryos were rinsed once in PBS-T following Proteinase K treatment and immediately fixed in 4% PFA for 20 minutes; 5) Following color reaction in BM Purple (Roche), embryos were washed three times in 1X PBS and immediately processed for histology and immunostaining.
4
Real-Time RT-PCR Analysis of HNSCC
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For real-time reverse transcriptase PCR (rtPCR) analysis, the RNA was isolated from all 149 HNSCC cases. For RNA isolation, 139 FFPE HNSCC specimens were dewaxed and 10 fresh-frozen HNSCC specimens were homogenized in Proteinase K buffer (50 mM Tris and 1 mM EDTA diluted in water). After dewaxing respectively homogenization the samples were digested with 80 μL Proteinase K (Roche Diagnostics GmbH, Unterhaching, Germany), 200 μL Proteinase K buffer plus Tween-20 (Tween 20 25% one part added to fifty parts of Proteinase K buffer) and 32 μL 10% sodiumdodecylsulfate overnight at 55°C. The next day, another 10 μL Proteinase K was added for further incubation overnight. Following digestion, the RNA isolation was continued with the InviTrap Spin Universal RNA Mini Kit (Stratec, Birkenfeld, Germany) according to the manufacturer's protocol. Finally, the RNA concentration of the samples was measured with the NanoDrop 1000 system (PEQLAB, Erlangen, Germany) and the samples were processed immediately or stored at −20°C until further processing. Only RNA probes with an Absorbance260/280 between 1.5 and 2.2 and a minimal RNA concentration of 5 ng/μL were used. The cDNA was then synthesized using Maxima® reverse transcriptase (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer's protocol.
5
RNA Purification from AGO Complexes
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Bead complexes were incubated in 100 μl of elution buffer (100 mM NaHCO3, 1% SDS) for 15 minutes at 25°C on a 1,400 rpm shaker. After spinning at 1,000g for 1 minute, the elution buffer was transferred to a new tube. An additional 100 μl of elution buffer was added to the bead complexes, elution was repeated and the eluates were then combined. To improve RNA phase separation of crosslinked RNA-AGO complexes, proteins were digested using proteinase K. 10 μl of proteinase K (Roche, catalog no. 03115887001) in 40 μl of proteinase K buffer (100 mM Tris-HCl (pH 7.5), 50 mM NaCl, 10 mM EDTA) was added to the 200 μl of eluate and samples were incubated at 37°C for 20 min. Samples were then subjected to phenol-chloroform extraction to purify the RNA.
6
TUNEL Assay for Apoptosis Detection
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TUNEL kits were purchased from Roche company. The paraffin sections were dewaxed by xylene at 60°C twice (each for 5 min), and dehydrated by gradient ethanol, then rinsed by phosphate-buffered saline (PBS) for 3 times (each for 5 min). The proteinase K solution was prepared (98 μL PBS and 2 μL proteinase K (Roche)), and each sample was appended with 100 proteinase K solution at 37°C for 30 min, then blocked for 10 min. After permeabilized by 1 μL 0.1% Triton X 100 and 0.1% sodium citrate solution on ice, the sections were supplemented with TUNEL reaction solution (Roche, PBS was used to replace the reaction solution in the NC group), transfected by peroxidase, and developed by diaminobenzidine (ZSGB-Bio, Beijing, China), and then counterstained by hematoxylin. Next, the sections were dehydrated, permeabilized, sealed, and observed by a light microscope.
7
Tumor Microdissection and DNA Extraction
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Microdissection of formalin-fixed, paraffin-embedded tumor and non-tumor mucosal sections was performed on hematoxylin-stained slides. The tumor and non-tumor mucosal components were microdissected separately and incubated in 50 μL buffer (0.5% Tween-20 [Boehringer Mannheim, Ingelheim, Germany], 20 μg proteinase K [Boehringer Mannheim], 50 mM Trizma base, pH 8.9, and 2 mM ethylenediaminetetraacetic acid) at 56 °C for 12–18 h. proteinase K was inactivated by incubating the samples at 100 °C for 10 min. All tumor samples in which the neoplastic cells accounted for at least 50% of the cell population were evaluated.
8
Proteinase K Protection Assays of Mitosomal Proteins
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proteinase K protection assays were performed on the 25,000g (mitosomal-enriched) pellet of infected RK-13 cells obtained as described above. The 25,000g pellet was washed twice with HSDP buffer without proteinase inhibitors and incubated with 50 μg ml−1 proteinase K (Roche) for 20 min, or 0.2% (v/v) Triton X-100 for 10 min at room temperature followed by incubation with 50 μg ml−1 proteinase K (Roche) for 20 min. The membrane fractions were then incubated with trichloroacetic acid at 20% (v/v) final concentration for 30 min on ice. Precipitated proteins were sedimented by centrifugation and solubilized in loading buffer. The mitosomal proteins were subsequently analysed by western blot.
9
Genotyping Prmt1 Knockout Mice
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Prmt1 KO mice were genotyped using their finger and tail tissues. To prepare the genomic DNA, finger and tail pieces were suspended in 40 μL of digestion buffer [1× modified Gitschier buffer; 67 mM Tris (pH 8.8), 16.6 mM ammonium sulphate ((NH4)2SO4), 6.7 mM magnesium chloride (MgCl2), 1% β-mercaptoethanol, and 0.5% Triton X-100] and boiled for 5 min. Samples were allowed to cool down and proteinase K (Roche, Mannheim, Germany) was added to a final concentration of 1 mg/mL. Then the samples were incubated at 55 °C for 1 h. Finally, the samples were boiled for 5 min to denature proteinase K. Thereafter, the samples were cooled down and 1 μL of total genomic DNA was used for PCR. Primers (Table S2 ) were used to detect the PRMT1 deletion by Ngn3-Cre.
10
TUNEL Assay for Cell Death Detection
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TUNEL assays were performed on slides treated for 22 min with Proteinase K (Roche) at 37 °C (10 ug/ml Proteinase K, 10 mM Tris-HCl pH 7.5). Staining was carried out using the In situ Cell Death Detection Kit, TMR Red (Roche).
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