| Description | HiFi II M-MLV (H -) is a reverse transcriptase that recombines and expresses mutated M-MLV genes using E. coli engineering bacteria. This enzyme can catalyze complementary DNA polymerization reactions using RNA or DNA: RNA hybrid strands as templates. The mutated HiFi II M-MLV (H -) reverse HiFi II M-MLV (H -) is a reverse transcriptase that recombines and expresses mutated M-MLV genes using E. coli engineering bacteria. This enzyme can catalyze complementary DNA polymerization reactions using RNA or DNA: RNA hybrid strands as templates. The mutated HiFi II M-MLV (H -) reverse transcriptase RNase H activity is missing, reducing RNA degradation in reverse transcription reactions and making it easier to obtain full-length cDNA. HiFi II M-MLV (H -) reverse transcriptase can synthesize the first strand of cDNA at 55 ℃, providing higher specificity, strong stability, and can synthesize up to 12 kb of cDNA with high cDNA yield. Suitable for the synthesis of first stranded cDNA, RT PCR, RT qPCR, and construction of full-length cDNA libraries.H665664Component10 KUStorageH665664AHiFi II M-MLV(H-) (200 U/µL) 50 µL-20℃. Avoid freeze/thaw cycle.H665664B5×SuperRT Buffer 1 mL-20℃. Avoid freeze/thaw cycle. Activity definition:Using Poly (A) as a template and oligo (dT) as a primer, the enzyme required to catalyze the addition of 1 nmol of dTTP within 10 minutes at 37 ℃ is defined as one active unit (U).Quality control:200 U of this enzyme reacted with 1 µ g of 16 S, 23 S rRNA at 37 ℃ for 1 hour, and the electrophoresis band of the RNA remained unchanged.Notes:1. During the operation process, RNase contamination should be avoided to prevent RNA degradation or cross contamination during experiments. It is recommended to perform RNA operations in specialized areas, use specialized instruments and consumables, and have operators wear masks and disposable gloves, and frequently change gloves.2. Disposable plastic containers should be used as much as possible for experiments. If glass containers are used, they should be treated with a 0.1% DEPC (diethyl pyrocarbonate) aqueous solution at 37 ℃ for 12 hours, and sterilized under high pressure at 120 ℃ for 30 minutes before use. Alternatively, glass containers should be sterilized under dry heat at 180 ℃ for 60 minutes before use. The sterile water used in the experiment should be treated with 0.1% DEPC and then subjected to high-pressure sterilization.3. All reagents in this reagent kit should be gently mixed upside down before use, avoiding foaming as much as possible, and used after brief centrifugation. The enzymes involved should be returned to -20 ℃ as soon as possible after use to avoid repeated freeze-thaw cycles.If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNAsin). This kit is not provided.Usage:Attention: 10 ng-5 µ G Total RNA can establish 20 µ L reaction system, if the total RNA amount is greater than 5 µ g. Please expand the reaction system proportionally.i Steps for reverse transcription:1. Dissolve the RNA template, primers, dNTP Mix, SuperRT Buffer, HiFi II M-MLV (H -), and RNase Free Water and place them on ice for later use.2. Prepare a reaction system according to the following table, with a total volume of 20 µ L. Reagent 20 µlReaction system Final concentration dNTP Mix，2.5 mM Each 4 µl 500 µM Each Oligo-dT Primer，100 µ MOr Random Primers ，50 µ Mor Specific Primer, 10 µ M 1 µl / RNA Template X µl 1 ng-5 µg 5×SuperRT Buffer 4 µl 1 × HiFi II M-MLV(H-) (200U /µL) 0.5-1 µL / RNase-Free Water up to 20 µL / Note: If the initial amount of RNA is less than 50ng, it is recommended to add RNA enzyme inhibitors (RNasins). This kit is not provided.3. Vortex shake and mix well, briefly centrifuge to collect the solution on the pipe wall to the bottom of the pipe. 4. Incubate at 55 ℃ for 1-30 minutes, and incubate at 85 ℃ for 5 minutes. After the reaction is complete, centrifuge briefly and cool on ice.5. Reverse transcripts can be directly used for PCR reactions and fluorescence quantitative PCR reactions, or stored at -20 ℃ for a long time.ii If the reverse transcription efficiency is low, or the RNA template secondary structure is complex and the GC content is high, the following steps are recommended:1. Dissolve the RNA template, primers, dNTP Mix, SuperRT Buffer, HiFi II M-MLV (H -), and RNase Free Water and place them on ice for later use.2. Prepare a reaction system according to the following table, with a total volume of 15 µ L. Reagent 20 µlReaction system Final concentration dNTP Mix，2.5 mM Each 4 µl 500 µM Each Oligo-dT Primer，100 µ MOr Random Primers ，50 µ Mor Specific Primer, 10 µ M 1 µl / RNA Template X µl 1 ng-5 µg RNase-Free Water up to 15 µL / 3. Incubate at 70 ℃ for 10 minutes and quickly ice bath for 2 minutes.4. Centrifuge briefly to collect the solution on the tube wall to the bottom of the tube.5. Add 4 to the above reaction solution µ L 5 x SuperRT Buffer.Note: If the initial amount of RNA is less than 50 ng, it is recommended to add RNA enzyme inhibitors (RNasins). This kit is not provided.6. Gently blow and mix well. If the reverse transcription primer is Oligo dT Primer or Specific Primer,7. Incubate at 42 ℃ for 2 minutes; If the reverse transcription primer is Random Primers, incubate at 25 ℃ for 10 minutes.8. Join 1 µ L HiFi II M-MLV (H -) (200 U/ µ L) Gently pat and mix well. Incubate at 55 ℃ for 50 minutes. Incubate at 85 ℃ for 5 minutes. After the reaction is complete, centrifuge briefly and cool on ice.9. Reverse transcripts can be directly used for PCR reactions and fluorescence quantitative PCR reactions, or stored at -20 ℃ for a long time... Read More | Source: Microorganism Isoelectric point: 6.5 Michaelis constant: 9.2×10^-3 M (D-Glucose); 8.6×10^-3 M (NAD) Optimum pH: 9.0~9.5 Fig. 1Optimum temperature: 55℃ Fig. 3pH Stability: 6.0-10.0 (25℃, 24hr) Fig. 2Thermal stability: <50℃ (pH 8.0, Source: Microorganism Isoelectric point: 6.5 Michaelis constant: 9.2×10^-3 M (D-Glucose); 8.6×10^-3 M (NAD) Optimum pH: 9.0~9.5 Fig. 1Optimum temperature: 55℃ Fig. 3pH Stability: 6.0-10.0 (25℃, 24hr) Fig. 2Thermal stability: <50℃ (pH 8.0, 30min) Fig. 4Inhibitors: NEM,SDS Effect of various chemicals: Table 1Reaction:... Read More | Biochemical Test:SDS-PAGE (purity > 80%); Western blot with patient sample.Calculated Isoelectric Point:pH 6.64 | Purity>95% SDS-PAGEFunctionLipid transport protein in adipocytes. Binds both long chain fatty acids and retinoic acid. Delivers long-chain fatty acids and retinoic acid to their cognate receptors in the nucleus | Purity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description： 100B, previously called S100 beta, belongs to the S100 family within the EF-hand superfamily of Ca2+ binding proteins. S100 proteins contain two EF-hand motifs that differ in affinity, separated by a hingePurity:>95%, by SDS-PAGE visualized with Coomassie® Blue Staining. Description： 100B, previously called S100 beta, belongs to the S100 family within the EF-hand superfamily of Ca2+ binding proteins. S100 proteins contain two EF-hand motifs that differ in affinity, separated by a hinge region with a hydrophobic cleft that is exposed upon Ca2+ binding. S100B is a 91 amino acid (aa) protein, after removal of the initial methionine, and is found as homodimers of 10.4 kDa monomers. Human S100B shares 99%, 98%, 100%, 99% and 97% aa sequence identity with mouse, rat, rabbit, equine and bovine S100B, respectively. Within the S100 family, human S100B shows the highest aa identity (59%) with S100A1. S100B is expressed primarily by astrocytes and oligodendrocytes in the central nervous system, and by Schwann cells in the peripheral nervous system. Ca2+-bound S100B interacts in vitro with at least 20 cytoplasmic proteins, including several structural molecules such as tubulin and GFAP. It can inhibit the phosphorylation of these kinase substrates and others such as tau and neuromodulin. Astrocytes can secrete S100B, which then acts in a cytokine-like manner. Nanomolar concentrations of S100B are secreted constitutively, promote proliferation, and are neurotrophic and anti-apoptotic. Blood levels of S100B reflect extracellular concentrations within the nervous system, and are elevated in Down’s syndrome, Alzheimer’s disease and Tourette’s syndrome, metabolic stress, acute brain injury and brain tumors. Micromolar concentrations of S100B can be destructive and pro-apoptotic; they induce the expression of iNOS, COX-2, IL-1, IL‑6 and TNF-alpha by microglia, astrocytes or neurons. Most extracellular actions of S100B can be mediated by RAGE (receptor for advanced glycation end products), which is also a receptor for other S100 proteins... Read More |