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Proteinase K from Tritirachium album belongs to the familiy of subtilisin-like serine proteases. It has an endo- and exoproteolytic activity. Activated by calcium (1 - 5mM), Proteinase K exhibits a broad substrate specificity. It degrades many proteins in the native state even in the presence of detergents after hydrophobic amino acids. Proteins will be completely digested, if the incubation time is long and the protease concentration high enough.
Inhibition/Inhibitors of Proteinase K
As Calcium is not directly involved in the catalytic process, removal of the Calcium ions will almost not influence the enzymatic activity but the stability of the enzyme is reduced (about 80% residual activity). The pH-optimum is at 8, but the enzyme is active over a wide pH-range (pH5 - 12). An elevation of the reaction temperature from 37°C to 50 - 60°C may increase the activity several times, like the addition of 0.5 - 1% SDS. Temperatures above 65°C, trichloroacetic acid or the serine protease inhibitors AEBSF (M6360) >, PMSF (M3194) > or DFP inhibit the activity.
Proteinase K will not be inhibited by EDTA, urea (1-4 M), SDS, citrate, iodoacetic acid or, interestingly, by other serine protease inhibitors like TLCK (M3375) > and TPCK (M3374) >. In case that proteinase K has to be inactivated, make sure, that the temperature is not below 95°C and the time not shorter than 10 minutes. A TCA-precipitation is well suited too.
Proteinase K is used for the digestion of proteins in cell lysates (tissue, cell culture cells) and for the release of nucleic acids, since it very effectively inactivates DNases and RNases. The digest with Proteinase K for the purification of nucleic acids is performed in the presence of EDTA (inhibition of magnesium-dependent enzymes).
Appearance: white lyophilisate
Activity: >30 AnsonU/mg protein (>30 units/mg)
DNAses and RNAses: not detectable
Solubility: easily soluble in water
pH (1% H2O, 20°C): 6.2 - 6.8
MW = ca. 27,000 g/mol.
application:Protein digest in DNA samples Isolation of genomic DNA from mouse tails Isolation of genomic DNA from cell cultures
Unit Definition:Activity: >30 U/mg lyophilizate. Specific activity: >40 U/mg protein. One unit is definded as the enzyme acitivity which liberates folin-positive amino acids and peptides corresponding to 1 µmol tyrosine in 1 minute at 37°C using haemoglobin as substrate.
Sicherheits Hinweise / SafetyH-Sätze: H315, H319 ,H334, H335
GHS-Symbole: GHS07, GHS08
Klassifizierungen / ClassificationEC-Nr: 254-457-8
Dokumente - Protokolle - Downloads
Here you will find information and further literature on Proteinase K Powder, usable for PCR. For further documents (certificates with additional lot numbers, safety data sheets in other languages, further product information) please contact Genaxxon biosience at: email@example.com or phone: +49 731 3608 123.
Dokumente - Protokolle - Downloads
dissolve Proteinase K (20mg/mL) in water or in 50mM Tris/HCl (pH8.0), 1.5mM Ca-Acetate.
Following some application examples based on 20mg/mL concentrations of Proteinase K dissolved in pure water or 50mM Tris, pH8.0.
Standard reaction buffer:
Cell lysis: 50 mM Tris-HCl (pH 7.5); 5 mM CaCl2; 0.5 % SDS.
DNA purification: 100 mM Tris-HCl (pH 8.0); 50 mM EDTA, 500 mM NaCl.
Some Application Protocols:
• Isolation of genomic DNA from mammal cells
(3h, 50°C) 10mM Tris/HCl (pH8.0), 100mM EDTA (pH8.0), 50mM NaCl, 0.5%SDS, 20µg/mL DNase free RNase, 100µg/mL proteinase K.
• Isolation of genomic DNA from mice tails
(overnight, 55°C) 20mM Tris/HCl (pH8.0), 5mM EDTA (pH8.0), 400mM NaCl, 1% SDS, 400µg/mL proteinase K.
• Rapid isolation of genomic DNA as a PCR-template from cell cultures
(1h, 37 °C) 67mM Tris/HCl (pH8.8), 16.6mM ammonium sulfate, 5mM β-mercaptoethanol, 6.7mM MgCl2, 6.7µM EDTA (pH8.0), 1.7µM SDS, 50µg/mL proteinase K.
• Preparation of DNA for PFGE, lysis in agarose block
(approx. 20h, 50°C). 100mM EDTA (pH8.0), 1 mM Tris/HCl (pH7.6), 20mM NaCl, 1% sarcosyl, 100µg/mL proteinase K.
• Purification of mRNA prior to cDNA synthesis (leads often to an increase in cDNA yield)
(1-2h, 37°C) 100mM Tris/HCl (pH7.5), 10mM EDTA (pH8.0), 50mM NaCl, 0.1% SDS, 5µg/mL proteinase K.
• Purification of PCR-reactions prior to cloning
(overnight, 55°C) PCR-reaction + 10mM Tris/HCl (pH8.0), 5mM EDTA (pH8.0), 400mM NaCl, 1% SDS, 400µg/mL proteinase K.
• Purification of plasmid-DNA prior to in vitro transcription
(1h, 37°C) Plasmid-DNA + 21mM Tris/HCl (pH8.0), 5mM EDTA (pH8.0), 50mM NaCl, 0.5% SDS, 100µg/mL proteinase K.
• RNase inactivation in ribonuclease-protection-assays
(30 min, 37°C) 30µL RNA/RNA-hybridisation mix + 300µL RNase digestion mix + 0.6% SDS, 300µg/mL proteinase K.
• Inactivation of DNase in transcription-run-on-assays
(30 min, 42°C) Suspension of nuclei in 36mM Tris/HCl (pH7.4), 17mM MgCl2, 0.7mM CaCl2, 170mM NaCl, 13mM EDTA (pH8.0), 0.5% SDS with DNase, 100µg/mL proteinase K.
• Denaturation of alkaline phosphatase when cipping vector DNA
(30 min, 56°C) 1 x dephosphorylation buffer + 0.5% SDS, 5mM EDTA (pH8.0), 100µg/mL proteinase K.
Hier finden Sie Artikel und Literaturzitate, in denen die Autoren auf die hohe Qualität dieses Genaxxonprodukts vertrauen.
Listed below are articles and references, in which the authors trust in the high quality of this Genaxxon product.
Decreased Enhancer-Promoter Proximity Accompanying Enhancer Activation
Nezha S. Benabdallah, Iain Williamson, Robert S. Illingworth, Lauren Kane, Shelagh Boyle, Dipta Sengupta, Graeme R. Grimes, Pierre Therizols, Wendy A. Bickmore
Mol Cell. 2019 Nov 7; 76(3): 473–484.e7. doi: 10.1016/j.molcel.2019.07.038
Glucocorticoid Receptor Binding Induces Rapid and Prolonged Large-Scale Chromatin Decompaction at Multiple Target Loci
Alasdair W. Jubb, Shelagh Boyle, David A. Hume, Wendy A. Bickmore
Cell Rep. 2017 Dec 12; 21(11): 3022–3031. Published online 2017 Dec 12. doi: 10.1016/j.celrep.2017.11.053
Enhancer turnover is associated with a divergent transcriptional response to glucocorticoid in mouse and human macrophages
Alasdair W Jubb, Robert S Young, David A Hume, Wendy A Bickmore
J Immunol. Author manuscript; available in PMC 2016 Jul 15.Published in final edited form as: J Immunol. 2016 Jan 15; 196(2): 813–822. Published online 2015 Dec 9. doi: 10.4049/jimmunol.1502009
Active promoters give rise to false positive ‘Phantom Peaks’ in ChIP-seq experiments
Dhawal Jain, Sandro Baldi, Angelika Zabel, Tobias Straub, Peter B. Becker
Nucleic Acids Res. 2015 Aug 18; 43(14): 6959–6968. Published online 2015 Jun 27. doi: 10.1093/nar/gkv637
ISWI Remodelling of Physiological Chromatin Fibres Acetylated at Lysine 16 of Histone H4
Henrike Klinker, Felix Mueller-Planitz, Renliang Yang, Ignasi Forné, Chuan-Fa Liu, Lars Nordenskiöld, Peter B. Becker
PLoS One. 2014; 9(2): e88411. Published online 2014 Feb 6. doi: 10.1371/journal.pone.0088411
PRC1 and PRC2 Are Not Required for Targeting of H2A.Z to Developmental Genes in Embryonic Stem Cells
Robert S. Illingworth, Catherine H. Botting, Graeme R. Grimes, Wendy A. Bickmore, Ragnhild Eskeland
PLoS One. 2012; 7(4): e34848. Published online 2012 Apr 9. doi: 10.1371/journal.pone.0034848
HP1 Binding to Chromatin Methylated at H3K9 Is Enhanced by Auxiliary Factors
Ragnhild Eskeland, Anton Eberharter, Axel Imhof
Mol Cell Biol. 2007 Jan; 27(2): 453–465. Published online 2006 Nov 13. doi: 10.1128/MCB.01576-06