Water-soluble, substrate for sortase mediated labeling of proteins. Sortase catalyzes a transpeptidase reaction between a specific internal sequence of a protein and an amine group present on the N-terminus of triglycine recently has become an area of great interest. This method of labeling proteins has been denoted as “Sortagging”. Proteins conjugated to DBCO-Gly-Gly-Gly can be further modified with azide-containing molecules creating site-specific protein conjugates. Examples of creating protein conjugates using sortagging include site-specifically PEGylating proteins,1 site-specific protein-lipid conjugates,2 and constructing peptides and glycosylphosphatidylinositol chimeras.3 Sortase has also been used in peptide synthesis to cyclize peptides to create macrocyclic peptides, glycopeptides4 and protein−protein conjugates.
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Water-soluble, substrate for sortase mediated labeling of proteins. Sortase catalyzes a transpeptidase reaction between a specific internal sequence of a protein and an amine group present on the N-terminus of triglycine recently has become an area of great interest. This method of labeling proteins has been denoted as “Sortagging”. Proteins conjugated to DBCO-Gly-Gly-Gly can be further modified with azide-containing molecules creating site-specific protein conjugates. Examples of creating protein conjugates using sortagging include site-specifically PEGylating proteins,1 site-specific protein-lipid conjugates,2 and constructing peptides and glycosylphosphatidylinositol chimeras.3 Sortase has also been used in peptide synthesis to cyclize peptides to create macrocyclic peptides, glycopeptides4 and protein−protein conjugates.
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Stool Total RNA Purification Kit
Product Info
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Product Info
Overview
Simultaneous isolation of both host RNA and microbial RNA (universal protocol)
Isolate full diversity of RNA from large RNA down to small and microRNAs
Eliminates PCR inhibitors including humic acids
High quality RNA for sensitive downstream application
Purification is based on spin column chromatography that uses Norgen’s proprietary resin separation matrix
This kit provides a convenient and rapid method to purify total RNA from small amounts of stool samples. All types of stool samples can be processed with this kit, including animal fecal samples, manure and samples collected using Norgen’s Stool Nucleic Acid Collection and Preservation Tubes (Cat. 45660). The kit removes all traces of humic acids using rapid and simple spin column procedures. Bead tubes are also provided for effective homogenization of stool. The kit purifies all sizes of RNA, from large mRNA and ribosomal RNA down to microRNA and small interfering RNA. Both host and microbial RNA is recovered. The protocol does not rely on the use of phenol or chloroform, thereby providing a user friendly procedure and allowing high-throughput analysis. The purified RNA is of the highest integrity, and can be used in a number of downstream applications including real time PCR and reverse transcription PCR for gene expression analysis. The procedure can be completed in approximately 30 minutes for 10 samples.
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Extracting Biological Insights from Stool
Tips and tricks for isolating high yield and quality DNA, RNA, miRNA and EV’s from fecal samplesDownload for Free
200 mg (fresh/frozen stool) or 400 μL (preserved stool)
Type of Stool Processed
Preserved, fresh, and frozen stool from humans and animals
Maximum Column Binding Capacity
50 μg
Maximum Column Loading Volume
600 μL
Time to Complete 10 Purifications
30 minutes
Storage Conditions and Product Stability All solutions should be kept tightly sealed and stored at room temperature. This kit is stable for 2 years after the date of shipment.
HL-SAN efficiently removes nucleic acids from buffers typically used in protein purification. Due to its high salt tolerance, it is the obvious choice for host-cell DNA removal in settings where salt is added to reduce aggregation. Especially efficient for removing nucleic acids from proteins with high affinity for DNA and RNA. Proven performance during lysis and early stages of protein purification processes, as well as high-salt eluates. Cold-adapted enzyme with excellent performance also at ambient temperatures and during over-night digestion at 4°C.
Optimum activity at high salt concentration (0.5 M NaCl)
Active at low temperatures (20% at 6ºC)
Easily inactivated
Broad pH range
Temperature stable
Figures
Figure 1. Optimum activity in solutions with high salinity
HL-SAN has optimum activity at ∼0.5 M NaCl, but operates at a broad range of [NaCl] and [KCl]. The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5, 5 mM MgCl2 with varying [NaCl] or [KCl]. The maximum activity was set to 100%.
Figure 2. Temperature and activity
HL-SAN has optimum activity at ~35°C, but works over a broad temperature range (20% activity at 10°C and 50°C). The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5 containing 5 mM MgCl2 and 0.5 M NaCl.
Fig 3. The effect of MgCl2 and MnCl2 concentration on the HL-SAN activity.
The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5, 0.5 M NaCl and with varying concentrations of MgCl2 or MnCl2. The activity of the sample containing 5 mM MgCl2 was set to 100%.
Figure 4. HL-SAN activity vs pH/[NaCl]
The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer with different pHs and different concentrations of NaCl. All buffers contained 5 mM MgCl2. The nature of the buffer was pH-dependent, but generally the NaCl-optimum was the same in all buffers/pHs. The exception was etanolaminbuffer at pH 9 and pH 9.5 in which the NaCl-optimum was shifted to the left (not shown).
Without NaCl, the specificity towards ssDNA and dsDNA is similar. At 0.5 M NaCl, the activity towards dsDNA increases, while the activity towards ssDNA is unaffected.
Figure 6. HL-SAN digests ssDNA to ~5-13 nt, and dsDNA to ~5-7 nt
The size of the end products from ssDNA varies from ~5-13 nt, while dsDNA is digested to around ~5-7 nt. The size of the end products seems to depend on the DNA sequence. Substrates 1 and 2 were ssDNA with different sequences and substrates 3 and 4 were dsDNA with similar sequences but with a FAM-label at different ends. Substrate 5 was dsDNA with the same sequence as substrate 3 and 4 but with a FAM-label at both ends.
Figure 7. HL-SAN activity decreases with increasing concentrations of glycerol
The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5, 5 mM MgCl2, 0.5 M NaCl and with increasing concentrations of glycerol. The activity of the control not containing glycerol was set to 100%.
Figure 8. The activity of HL-SAN at different concentrations of imidazole
The activity of HL-SAN was tested in a 25 mM Tris-HCl buffer, pH 8.5, 5 mM MgCl2, 0.5 M NaCl and with varying concentrations of imidazole. The activity of the control not containing imidazole was set to 100%.
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HL-SAN efficiently removes nucleic acids from buffers typically used in protein purification. Due to its high salt tolerance, it is the obvious choice for host-cell DNA removal in settings where salt is added to reduce aggregation. Especially efficient for removing nucleic acids from proteins with high affinity for DNA and RNA. Proven performance during lysis and early stages of protein purification processes, as well as high-salt eluates. Cold-adapted enzyme with excellent performance also at ambient temperatures and during over-night digestion at 4°C.
Our SNPsig® kits use our own proprietary genotyping method to enable the identification of SARS-CoV-2 variants of concern. These products can be used on any real-time PCR machine using familiar protocols, whilst resulting in exceptional genotyping data.
Positive control templates for wild-type and variants are supplied in every kit to make data interpretation simple.
Our SNPsig® technology provides an alternative to sequencing as well as S gene target failure (SGTF) that enables scientists to analyse and monitor these specific genomic mutations. Our kits can provide a pivotal role in screening for SARS-CoV-2 variants for the purpose of genomic surveillance and studies.
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For the detection of the SARS-CoV-2 (20J/501Y.V3 Brazil) Rapid detection of specific detection profiles High priming efficiency Sensitive to < 100 copies of target
Positive copy number standard curve for quantification
Accurate controls to confirm findings
96 reactions, includes master mix
