Proteinase k

Manufactured by Wisent

Proteinase K is a serine protease that is commonly used in molecular biology and biochemistry for the digestion and degradation of proteins. It is an enzyme that catalyzes the hydrolysis of peptide bonds in a wide range of proteins, making it a versatile tool for various applications.

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Lab products found in correlation

Directpcr lysis reagent, by Viagen Biotech (2 mentions) Facsaria 3, by BD (2 mentions) Nanodrop 2000c, by Thermo Fisher Scientific (2 mentions) Proteinase k, by Thermo Fisher Scientific (1 mentions) Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions) Tris cl, by Wisent (1 mentions) Sodium dodecyl sulfate (sds), by Roche (1 mentions) Mgcl2, by Thermo Fisher Scientific (1 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions) Lightcycler 480 2, by Roche (1 mentions) Tissuelyser lt, by Qiagen (1 mentions) Qiazol, by Qiagen (1 mentions) Proteinase k, by Qiagen (1 mentions) Quantitect reverse transcription, by Qiagen (1 mentions)

3 protocols using proteinase k

Flow Cytometric Analysis of HIV p24+ T Cells

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The frequency of p24 double positive cells (KC57+, 28B7+) was determined by flow cytometry in gated viable CD8-CD45RA- T cells. An example of the gating strategy is represented in Supplementary Fig. 7a, b. In all experiments, CD4 + T cells from an HIV-uninfected control were included to set the threshold of positivity. Single p24 double positive (p24+ cells) and double negative cells (p24- cells) were indexed-sorted on a BD FACS ARIA III. Cells were sorted in 96-wells PCR plates containing 7.6 μL of DirectPCR Lysis Reagent (Viagen Biotech) and 0.4 μL of 10 mg/mL proteinase K (from Wisent, 25530–015). The PCR plates were subsequently incubated at 55 °C for 1 h for cell lysis followed by 15 min at 85 °C to inactivate proteinase K. Index-sorting data of p24+ cells were analyzed using FlowJo version 10.5.3.

Gantner P., Pagliuzza A., Pardons M., Ramgopal M., Routy J.P., Fromentin R, & Chomont N. (2020). Single-cell TCR sequencing reveals phenotypically diverse clonally expanded cells harboring inducible HIV proviruses during ART. Nature Communications, 11, 4089.

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Sorting and Sequencing of HIV-Infected T Cells

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The frequency of p24 double positive cells (KC57⁺, 28B7⁺) was determined by flow cytometry in gated viable T cells. Examples of gating strategies are represented in Fig. S1 and S9. In all experiments, CD4⁺ T cells from an HIV-uninfected control were included to set the threshold of positivity. Single p24 double positive (p24⁺ cells) were indexed-sorted and p24 double negative (p24⁻ cells) were bulk sorted on a BD FACS ARIA III. Single-cells were sorted in 96-wells PCR plates containing 7.6μL of DirectPCR Lysis Reagent (Viagen Biotech, Cat#301-C) and 0.4 μL of 10mg/mL proteinase K (Wisent, Cat#25530–015). The PCR plates were subsequently incubated at 55°C for 1 hour for cell lysis followed by 10 min at 95°C to inactivate proteinase K. Bulk-sorted and pelleted cells were digested in 30μL of proteinase K (Cat#25530-015, Invitrogen; final concentration 400 μg/ml, TriS HCl (10mM) and KCl (50nM) overnight at 55°C followed by 10 min at 95°C to inactivate proteinase K. Cell lysates were then used for TCR sequencing or near-full length HIV genome sequencing.

Derbyshire V., Wood D.W., Wu W., Dansereau J.T., Dalgaard J.Z, & Belfort M. (1997). Genetic definition of a protein-splicing domain: Functional mini-inteins support structure predictions and a model for intein evolution. Proceedings of the National Academy of Sciences of the United States of America, 94(21), 11466-11471.

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Comprehensive RNA Isolation and Analysis

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Total RNA was purified from mouse tibialis anterior, soleus, triceps, quadriceps, latissimus dorsi, diaphragm, heart, and brain after flash freezing in liquid nitrogen. The isolation process started with a crude homogenization of 25 mg of tissue in proteinase K solution (10 mM Tris-Cl, pH 7.5 [Wisent]; 10 mM EDTA [Sigma-Aldrich]; 2% SDS [Roche]; 500 mM NaCl [Fisher Scientific]; 1.5 mM MgCl2 [Fisher Scientific]; and 500 μg/mL proteinase K [QIAGEN]), followed by complete homogenization with a TissueLyser LT (QIAGEN) using a ball bearing. After a 30-min incubation at 55°C, standard QIAzol (QIAGEN) protocol for tissue extraction was performed along with RNA quantification with a NanoDrop 2000c (Thermo Scientific), and RNA integrity was verified on a 2100 Bioanalyzer (Agilent Technologies). cDNA was then produced using Quantitect Reverse Transcription (QIAGEN) on 200 ng of RNA, and RT-qPCR was used to determine mRNA levels for DMPK and Dmpk mRNA, with Hprt1, Rpl13a, and Tbp RNAs as normalization controls (primers in Table S3) on a LightCycler 480 II (Roche) using SYBR Green I Master hot start reaction mix.

Jauvin D., Chrétien J., Pandey S.K., Martineau L., Revillod L., Bassez G., Lachon A., McLeod A.R., Gourdon G., Wheeler T.M., Thornton C.A., Bennett C.F, & Puymirat J. (2017). Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice. Molecular Therapy. Nucleic Acids, 7, 465-474.

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