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Product information "Proteinase Low-Temp - solution (200 units/mL), easy to inactivate"
Genaxxon Proteinase Low-Temp is a nonspecific endopeptidase derived from an Arctic marine microbial source. It has a broad substrate specificity and is easy to inactivate after use. Starting at a temperature of 60°C, the Genaxxon Proteinase Low-Temp is heat-inactivated, making it ideal for heat-sensitive samples.
The Proteinase Low-Temp is most active in the temperature range of 25°C-40°C. Recommended inactivation conditions are: Typically up to 15 minutes at 40°C – 65°C (inactivation by denaturation).
It is known that histones and other proteins shield nucleic acids from an optimal interaction with other DNA-binding proteins and enzymes. Genaxxon Proteinase Low-Temp is ideal for transforming chromatin and other compact nucleic acids into mere DNA. The enzyme is easily inactivated. This allows thermal inactivation at temperatures that provide RNA integrity and avoid the dissociation of dsDNA.
Advantages:
- Easy to deactivate
- Active at high salt content
- Compatible with downstream analyzes
Application examples:
Preparation of chromosomal DNA for PFGE
- Degradation of nucleases (DNase, RNase) in the preparation of nucleic acids, including the recovery of native RNA
- Isolation of genomic DNA from tissue of mouse tails
- Isolation of genomic DNA from cell cultures
Specific activity: >30 U/mg. Foreign activity: RNase and DNase undetectable. Temperature optimum: + 25°C-40°C (pH 7-10). Recommended inactivation conditions: Typically up to 15 minutes at 40°C – 65°C (inactivation by denaturation).
Inhibitors: General Serine protease inhibitors such as PMSF (M3194) >.
Instructions for use: The enzyme is stable up to 2 years when stored at -20°C. Genaxxon Proteinase Low-Temp is stable even when stored at 4°C (>6 months) and room temperature (>4 weeks).
The optimal enzyme activity is between pH 7 and 10. Recommended temperature range for the activity is 25-40°C. The activity of Genaxxon Proteinase Low-Temp is not dependent on divalent cations such as calcium (Ca2 +) and is therefore active in buffers containing EDTA (<40°C reaction temperature).
Genaxxon Proteinase Low-Temp is suitable for the digestion of proteins in the presence of SDS (0.2-1%) and urea (1-5 M). The enzyme tolerates 500mM guanidine thiocyanate and 1% Triton X-100 (>50% activity). There is no significant loss of activity at 400 mM NaCl.
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).
Proteinase K can be obtained from Genaxxon as Powder (M3036) > or as 20mg/mL Solution (M3037) >.
- Source: Recombinantly produced in Pichia pastoris
- Specific activity: >30 Units/mg
- Concentration: min. 200 U/mL
- Size: 34 kDa
- Specificity: Nonspecific endopeptidase
- Activity:
- Highly active in temperature range of 25–40°C
- pH: 7–10 - Inactivation:
Typically up to 15 minutes at 40°C – 65°C (inactivation by denaturation). - Tolerance to typical buffer additives:
- SDS: 0.2–1%
- Urea: 1–5 mM - Inhibitors: The enzyme is inhibited by general serine protease inhibitors, such as PMSF.
- Storage: The enzyme is stable upon storage at - 20°C for up to 2 years. Genaxxon Proteinase is also stable upon storage at 4°C (> 6 months) and room temperature (> 4 weeks). Genaxxon Proteinase is stable at dilute concentrations (< 0.1 mg/ml) in solutions.
Application:
Protein digest in DNA samples for example: Typical uses will be direct-PCR settings of single and few cells. Isolation of vaccinia virus DNA. Purification of genomic DNA from bacteria (miniprep).Units:
One unit is defined as the amount of enzyme that produces one μmol 4-nitroaniline (extinction coefficient 8.8 mM-1 at 410 nm) at 25°C, pH 8.0 per minute (buffer conditions 50 mM Tris-HCl pH 8.0, 1% DMSO, 1 mM Suc-Ala-Ala-Pro-Phe-pNA).Source:
Pichia pastorisSafety Information:
Classification:
Documents:
Product descriptionThis enzyme differs from normal Proteinase K as it is easier to inactivate and gentler on downstream procedures. Its main purpose is to reduce chromatin to naked DNA prior to analysis. It is best fitted to analysis of small amounts of cells, where endogenous proteinases will not have an adverse effect on downstream analysis
Possible protocol for tissue cells:
Seed small amounts of cells in 96-well plate (few thousands?)
Following day – wash and lyse cells in lysis buffer supplemented with AZ Protease (7.5 U/mL)
Transfer 2µL lysate to a 10µL qPCR reaction kept on ice
Run qPCR directly
Heat inactivate might be necessary in some cases (e.g. in case of not working on ice, as this means that used enzymes will be more susceptible for protease activity), then a typical inactivation step might be up to 15 minutes at 40-65°C, depending on degree of inactivation needed.
Accessory Items
DNase I is an endonuclease isolated from bovine pancreas that digests double- and single-stranded DNA into oligo- and mono-nucleotides. DescriptionDNAse I is used, among other things, to avoid unwanted cell clumping during tissue disaggregation, which makes dispersion of individual cells difficult. Since tissue disaggregation and subsequent isolation of single cells is always accompanied by rupture and lysis of some cells, DNA is released from these cells into the surrounding medium. The released DNA in the medium is thought to be responsible for undesirable cell clumping during tissue dissociation procedures. This clumping effect can be avoided by adding deoxyribonuclease (DNase) to the dissociation medium and has been described for a variety of different cell and tissue types. DNase I is suitable for eliminating DNA from RNA preparations prior to sensitive applications, such as RT-PCR. Since no RNA purification procedure removes 100% of the DNA, RNA samples should be digested with DNase I before RT-PCR. A simple 15 minute digestion at room temperature removes the contaminating DNA. DNase I is inactivated by adding the stop solution and heating. Heating also denatures the RNA, so the RNA can be used directly for reverse transcription. This DNAse I is not RNase-free and has to be chemically treated if used for RNA isolation procedures.
Overview:Proteinase K is a robust and versatile serine protease used extensively in molecular biology for the digestion of proteins in nucleic acid preparations. This solution, at a concentration of 20 mg/mL, is ideal for a wide range of applications including the preparation of DNA and RNA samples. The PCR grade quality ensures that the enzyme is free from contaminants that could interfere with downstream applications, making it highly suitable for sensitive techniques such as PCR.Product Details:ready-to-useConcentration: 20 mg/mLactive over a wide range of reaction conditionsAn elevation of the reaction temperature from 37°C to 50 - 60°C may increase the activity several timesQuality: PCR grade, ensuring high purity and no interference with PCR reactions.Applications: Suitable for DNA and RNA extractionRemoval of protein contaminantsPreparation of samples for various molecular biology techniques.Benefits:High Purity: The PCR grade quality guarantees minimal contamination, ensuring reliable results in sensitive applications. Versatility: Ideal for various molecular biology procedures, including sample preparation for PCR.Convenience: Ready-to-use solution at an optimal concentration for ease of use. Notes:The recommended working concentration of Proteinase K is 0.05 to 1 mg/mL. The activity of the enzyme is stimulated by 0.2 to 1% SDS and also by 1 to 4M ureaCa2+ protects Proteinase K against autolysis and increases the thermal stabilityStable over a wide pH range: 4.0 to 12.5, optimum pH 7.5 to 8.0Activity optimum: 50°C to 55°CRapid denaturation of enzyme occurs at temperatures above 65°CFeatures:No detected exonuclease, endonuclease or RNase activitySpecific activity: >40 units/mg protein Activity: ≥30 units/mg lyophilizateSolubility: ≥20 mg/mLDNA content ≤10 pg/mgShipping conditions: Shipment on wet iceInhibitors of proteinase KWhich substances can inhibit proteinase K? Proteinase K can be inhibited by a variety of substances. These include ‘denaturing substances’, detergents and proteinase inhibitors. Sensitivity to inhibitors depends pretty much on the specific application (exact pH, concentration, temperature). Inhibitors frequently used in biochemistry and molecular biology are:SDS: High concentrations of SDS can denature and inactivate proteinase K.EDTA: EDTA is a chelating agent that can bind to metal ions that are essential for proteinase K activity.Urea: High concentrations of urea can denature and thereby inactivate proteinase K.Detergents: Some detergents, such as Triton X-100 or Tween® 20, can inhibit proteinase K, especially when the detergents are present in high concentrations.Protease inhibitors: Protease inhibitors, such as phenylmethylsulfonyl fluoride (PMSF), can irreversibly inhibit proteinase K.Chaotropic salts: Depending on the DNA extraction protocol, the presence of chaotropic salts or detergents may increase proteinase K activity. However, the same reagents may also inhibit proteinase K activity.Proteinase K can be obtained from Genaxxon as Powder (M3036) > or as 20mg/mL Solution (M3037) >.Produced by Genaxxon bioscience GmbH, founded in 2002 by Dr. Norbert Tröndle to provide reliable products for PCR and custom cell culture media formulations, the Proteinase K Solution 20 mg/mL offers a dependable and efficient solution for your molecular biology needs. Achieve consistent, high-quality results with an enzyme designed specifically for your requirements.
Protease inhibitor mix. Special mixture of 5 water-soluble protease inhibitors with broad specificity for the inhibition of cysteine-, serine- and metalloproteases. Recommended for general applications and where the use of organic solvents should be avoided. This mix contains AEBSF >, Aprotinin >, E-64 >, Leupeptin > and EDTA. The content of 1 vial dissolved in 1mL water results in a 100-fold concentrate suitable for the treatment of 100mL crude protein extract. Activity spectrumAEBSF is an inhibitor against Serine proteases. Aprotinin is an inhibitor against Serine proteases. E-64 is an inhibitor against Cysteine proteases. Leupeptin is an inhibitor against Cystein proteases and trypsin-like Serine proteases and EDTA as an inhibitor against Metallo proteases. More ready-to-use protease inhibitor mixes can be found here: - Protease-Inhibitor-Mix I > (general applications, contains no EDTA) - Protease-Inhibitor-Mix II > (general applications, contains EDTA)- Protease-Inhibitor-Mix III > (formulated for use with bacterial extracts, contains EDTA)- Protease-Inhibitor-Mix IV > formulated for use with plant extracts, contains no EDTA)- Protease-Inhibitor-Mix V > (formulated for use with mammalian extracts, contains no EDTA) You will find further protease inhibitors here: Inhibitors >: Pepstatin A >, AEBSF >, Leupeptin >, TLCK >, PMSF >, Bestatine > or Diisopropylphosphorofluoridat (DFP) >.
Ribonuclease A is an endo-ribonuclease specifically cleaving single stranded RNA at the 3' side of pyrimidine bases (cytosine and uracil). RNAse A ist used to prepare RNA-free DNA, to digest non-hybridised regions of RNA-DNA hybrids and as a molecular weight marker. The pH optimum for RNAse A is between 7.0-7.5. RNAse A is inhibited by Diethylpyrocarbonate (DEPC) >, guanidinium salt (4M GuaSCN), beta-Mercaptoethanol, heavy metals, vanadyl-ribonucleoside complexes, RNAse-inhibitor from human placenta and by competitive DNA. RNAse A cleaves single- and double-stranded RNA and RNA in RNA:DNA hybrids at low salt conditions (100 mM NaCl). At high salt conditions (>300 mM NaCl), RNAse A cleaves single-stranded RNA only. The enzyme can only be removed by Proteinase K digest and subsequent phenol extraction. Elimination of DNAse activity: Product S5231 (RNAse A (not certified DNAse free)) may contain DNAse activity. As RNAse A is heat stable it is recommended to heat inactivate DNAse activity before use. Procedure: 10mg/mL RNAse A in 0.01 sodium acetate (pH5.2) have to be heated to 100°C for 15 minutes in a water bath. After 15 minutes, the RNAse shall remain in the water bath while the water cools down to room temperature. pH can be adjusted by addition of 0.1 time the volume of 1M Tris-HCl (pH7.4). After aliquotation of the now DNAse free RNAse A solution, each aliquote should be stored a -20°C. NOTE: RNAse A precipitates if concentrated solutions are heated to 100°C at neutral pH! Stock solution: Stock solutions are prepared at concentrations from 1 - 10mg/mL in 10mM Tris/HCl, pH7.5; 15mM NaCl or in 10mM Tris/HCl, pH7.5; 1mM EDTA, pH8.0 (TE buffer).The recommended working concentration is 10µg/mL (removal of RNA from plasmid preparations; 1hr, RT) or 100ng/mL (preparation of 'blunt ends' of double-stranded cDNA). Stability:RNase A aggregates during lyophilizing and storage. It has a high affinity to glas surfaces, which has to be taken into consideration. At neutral pH (e. g. in PBS pH 7.4) and high concentrations (>10mg/mL) the enzyme will precipitate. At +2°C to +8°C (lyophilized) it is stable for several years (dry storage), in solution (-20°C) several years or (+2°C to +8°C) several weeks.