| Description | Product Introduction:Due to the low stoichiometry of phosphorylation, the concentration of phosphopeptides is a key step for the successful implementation of phosphoproteomics experiments. PolyMAC provides an effective and significantly improved method to achieve more complete enrichment of Product Introduction:Due to the low stoichiometry of phosphorylation, the concentration of phosphopeptides is a key step for the successful implementation of phosphoproteomics experiments. PolyMAC provides an effective and significantly improved method to achieve more complete enrichment of phosphopeptides. This highly selective enrichment method can be used for most complex samples because it offers optimal specificity and recovery rate. PolyMAC is a polymer-based immobilized metal ion affinity capture method with excellent phosphoproteome coverage and recovery rate. It is a soluble nanopolymer designed to interact with phosphopeptides in solution, and then be captured on a solid-phase carrier for washing and phosphopeptide elution. Compared with the commonly used titanium dioxide and immobilized metal ion affinity capture methods, PolyMAC has better reproducibility and enrichment of phosphorylated peptides, with the selectivity of phosphorylated peptides close to 95% and the recovery rate > 90%. Importantly, PolyMAC has very good recovery rate and selectivity for samples with low total protein phosphorylation levels.Experimental Flowchart of Phosphopeptide Protein (Peptide Fragment) Extraction Kit (P1456460)Product Components and Storage Conditions:P1456460Component24T48T96TStorageP1456460APolyMAC Beads0.75 mL1.5 mL3 mL4℃P1456460BPolyMAC Loading Buffer25 mL50 mL100 mLRTP1456460CPolyMAC Washing Buffer I25 mL50 mL100 mLRTP1456460DPolyMAC Washing Buffer II25 mL50 mL100 mLRTP1456460EPolyMAC Elution Buffer3.75 mL7.5 mL15 mLRTP1456460FPolyMAC Tips24T48T96TRTProduct Features:Easy to operate: Phosphopeptides can be prepared quickly, requiring only a metal bath and a conventional centrifuge.High stability: Strict quality inspection is conducted for each batch, ensuring high reproducibility of experimental results.Operating Procedure:1.Add 200 µL of Loading Buffer to the dried sample (50 µg - 100 µg) after enzymatic hydrolysis and desalting, and fully resuspend the sample.2.Vortex the PolyMAC Beads thoroughly (for 10 - 20 seconds), then take 25 µL into a 1.5 mL centrifuge tube, centrifuge instantaneously for 2 - 3 seconds, and then remove the upper storage solution. (The Beads settle quickly, so the operation should be fast when taking them.)3.Add the resuspended sample to the centrifuge tube containing PolyMAC Beads, then place it on a mixer, and shake vigorously at 26°C, >1200 rpm for 25 minutes.4.Add the sample to PolyMAC - Tips, first centrifuge at 20 g for 2 minutes, then centrifuge at 100 g for 1 minute to ensure that the sample solution flows out from the tip of the Tips into the centrifuge tube. (The rotation speed should not be too high, and the operation should be fast.)5.Wash once with 200 µL of Loading Buffer: first centrifuge at 20 g for 2 minutes, then centrifuge at 100 g for 1 minute, and discard the filtrate. Repeat this step 3 times. (The rotation speed should not be too high, and the operation should be fast.)6.Wash once with 200 µL of Washing Buffer I: first centrifuge at 20 g for 2 minutes, then centrifuge at 100 g for 1 minute, and discard the filtrate. Repeat this step 3 times. (The rotation speed should not be too high, and the operation should be fast.)7.Wash once with 200 µL of Washing Buffer II: first centrifuge at 20 g for 2 minutes, then centrifuge at 100 g for 1 minute, and discard the filtrate. Repeat this step 3 times. (The rotation speed should not be too high, and the operation should be fast.)8.Put the Tips into a new centrifuge tube to collect the eluted phosphopeptides. Add 50 µL of Elution Buffer to the Tips, centrifuge at 20 g for 2 minutes, then add another 50 µL of Elution Buffer, first centrifuge at 20 g for 2 minutes, then centrifuge at 100 g for 1 minute, and collect the filtrate. (The rotation speed should not be too high, and the operation should be fast.)Note: In steps 4 - 8, if the liquid flows down slowly, the rotation speed can be increased appropriately, gradually increasing to 50 g, and do not increase too much at one time!9.If there is any solution remaining in the pipette tip, push it out into the collection tube with a pipette, lyophilize it, and store it at -80°C for mass spectrometry detection.Precautions:This product is only for scientific research use by professionals, and shall not be used for clinical diagnosis or treatment, nor for food or drugs... Read More | Inquire | DescriptionUse in combination with the KitAlysis Bench Top Inertion Box (Z742064) or a glove box/glove bag to provide inert atmosphere for kit set-up.Designed to be used with KitAlysis High-Throughput Screening Kits.Components:24-Well Reaction BlockTorque ScrewdriverSmall screwdriver to easily DescriptionUse in combination with the KitAlysis Bench Top Inertion Box (Z742064) or a glove box/glove bag to provide inert atmosphere for kit set-up.Designed to be used with KitAlysis High-Throughput Screening Kits.Components:24-Well Reaction BlockTorque ScrewdriverSmall screwdriver to easily remove torqued screws after reaction is complete.10 Reaction Block Replacement Screws... Read More | Product introduction：This kit uses an improved SDS alkaline lysis method combined with DNA preparation membrane to selectively adsorb DNA to achieve the purpose of rapid purification of plasmid DNA. It is suitable for extracting up to 100u of high-purity plasmid DNA from 30-100 ml of Product introduction：This kit uses an improved SDS alkaline lysis method combined with DNA preparation membrane to selectively adsorb DNA to achieve the purpose of rapid purification of plasmid DNA. It is suitable for extracting up to 100u of high-purity plasmid DNA from 30-100 ml of bacterial culture for sequencing, in vitro transcription and translation, restriction enzyme digestion, bacterial transformation and other molecular biology experiments.Scope of application：Nucleic acid extraction and purification... Read More | Product contentU665751Component100 TStorageU665751A2×UltraSYBR One Step Buffer1.4 mL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751BUltraSYBR One Step EnzymeMix50 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751C50×High ROX50 µL-20℃. AvoidProduct contentU665751Component100 TStorageU665751A2×UltraSYBR One Step Buffer1.4 mL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751BUltraSYBR One Step EnzymeMix50 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751C50×High ROX50 µL-20℃. Avoid freeze/ Thaw cycle. Protect from light.U665751DRNase-Free Water1.5 mL-20℃. Avoid freeze/ Thaw cycle. Product Introduction This product is a specialized kit for one-step Real-Time RT-qPCR. The SYBR Green I fluorescent dye contained can bind to all double-stranded DNA, allowing this product to be used for the detection of many different target sequences without the need to synthesize specific labeling probes. Real Time RT-qPCR reaction using this product, reverse transcription and quantitative PCR are carried out in the same reaction system, there is no need to add reagents during the reaction, no need to open the cap of the tube, avoiding contamination while improving the efficiency of the experiment. The new high-efficiency reverse transcriptase RNase H is activity-deficient, which reduces the degradation of RNA in the reverse transcription reaction. The enzyme has high reverse transcription efficiency and can perform a good reverse transcription reaction on a small amount of RNA template. It has high affinity to RNA and can read through RNA templates with high GC content and complex secondary structure. New efficient hot start enzyme, the enzyme activity is closed at room temperature, thus effectively avoiding non-specific amplification caused by non-specific binding of primers and templates or primer dimerization at room temperature, which greatly improves the accuracy of fluorescence quantitative PCR reaction. The included buffer system maximizes the efficacy of both enzymes at the same time and improves efficiency. This product has high sensitivity, high specificity, wide linear range, and more accurate quantification of target genes.ROX dye is used to correct the fluorescence signal error generated between wells of a quantitative PCR instrument, and is generally used with Real Time PCR amplifiers from ABI, Stratagene, and other companies. The excitation optics vary from instrument to instrument, so the concentration of ROX dye must be matched to the corresponding fluorescence quantitative PCR instrument. Instruments that do not require ROX calibration (U665567) Roche LightCycler 480, Roche LightCyler 96, Bio-rad iCyler iQ, iQ5, CFX96 and others. Instruments that require High ROX calibration (U665751) ABI Prism 7000/7300/7700/7900, Eppendorf, ABI Step One/Step One Plus, and others.matters needing attention1. Before using the reagents in this kit, please mix them gently by turning them up and down to avoid foaming as much as possible, and use them after brief centrifugation.2. This product uses RNA as the template for one-step RT-PCR experiment, RNase contamination should be avoided during operation, it is recommended to operate RNA in a special area, use special instruments and consumables, the operator with a mask and disposable gloves and often change the gloves, the experiment-related consumables should be processed with 0.1% DEPC (diethyl ether of pyrocarbonate) aqueous solution at 37℃ for 12 hours and autoclaved for 30 minutes before use. Sterilize for 30 minutes before use.3. UltraSYBR One Step RT-qPCR Buffer contains SYBR Green I fluorescent dye. Avoid bright light when storing this product or preparing PCR reaction solutions.4. Repeated freezing and thawing of each reagent in this kit should be avoided; repeated freezing and thawing may degrade the product performance. This product can be stored for a long time at -20℃, protected from light. If frequent use is required in the short term, it can be stored at 2-8℃.5. This kit must use specific primers, the choice of primers can be selected according to specific experiments, the good or bad primer design directly affects the results of RT-PCR reaction, the design of primers need to consider the GC content, primer length, primer position, the secondary structure of the PCR product and other factors, it is recommended to use a professional primer design software for design.6. This product cannot be used for fluorescent quantitative PCR by the probe method.Usage1. Dissolve RNA template, primers, 2× UltraSYBR One Step Buffer, UltraSYBR One Step EnzymeMix and RNase-Free Water and set aside on ice.2. PCR reaction system:Reagents25 µl Reaction systemFinal concentration2×UltraSYBR One Step Buffer12.5 µl1×Forward Primer，10 µM0.5 µl0.2 µM¹⁾Reverse Primer，10 µM0.5 µl0.2 µM¹⁾UltraSYBR One Step EnzymeMix0.5 µl RNA TemplateX µl10 pg – 100 ng50×Low ROX or High ROX（optional）2）0.5 µl1×RNase-Free Waterup to 25 µlNote: 1) Usually, the primer concentration of 0.2µM can get better results, and the final concentration of 0.1-0.5µM can be used as a reference for setting the range. If the amplification efficiency is not high, the concentration of primer can be increased; when non-specific reaction occurs, the concentration of primer can be decreased, thus optimizing the reaction system.(2) The excitation optical system varies from instrument to instrument, choose to add 50×Low ROX or 50×High ROX according to the instrument using fluorescence quantification.3. Vortex and shake to mix, centrifuge briefly, and collect the solution at the bottom of the tube.4. RT-qPCR reaction conditions (fluorescence quantitative PCR is a two-step method), this program is based on the ABI 7500 fluorescence quantitative PCR instrument as an exampleNote: 1) It is recommended to use two-step PCR reaction program, if you improve the reaction specificity, you can increase the annealing temperature to 60-64 ℃ as a reference for the setting range; if you do not get good experimental results due to the use of primers with lower Tm values, etc., you can try to carry out three-step PCR amplification.(2) For melting curve analysis, please set up the program recommended by the fluorescence quantitative PCR instrument used, and this program is set up with the ABI 7500 fluorescence quantitative PCR instrument as a reference.RT-qPCR reaction conditions (fluorescence quantitative PCR was a three-step method):Note: 1) For three-step PCR amplification, please use the range of 56℃-64℃ as the setting reference for the annealing temperature.(2) For melting curve analysis, please set up the program recommended by the fluorescence quantitative PCR instrument you are using, this program is ABI750 fluorescent quantitative PCR instrument as a reference setting... Read More |