| Description | Product Introduction:This kit is used to enrich low-abundance proteins in plasma samples and rapidly prepare proteomic samples, and the processed samples can be used for subsequent mass spectrometry detection.Product Components and Storage Conditions:M1456374Component10T25TStorageM1456374 AMagnetic Product Introduction:This kit is used to enrich low-abundance proteins in plasma samples and rapidly prepare proteomic samples, and the processed samples can be used for subsequent mass spectrometry detection.Product Components and Storage Conditions:M1456374Component10T25TStorageM1456374 AMagnetic beads Max200 µL500 µL4℃M1456374 BIncubation Buffer I4.8 mL12 mLRTM1456374 CIncubation Buffer II6 mL15 mLRTM1456374 DWashing Buffer6 mL15 mLRTM1456374 ELysis Buffer0.6 mL1.5 mL-20℃. Store in the dark.M1456374 FBalance Buffer2.8mL7 mL4℃M1456374 GDigest Buffer16 µL40 µL-20℃M1456374 HStop Buffer300 µL750 µLRTM1456374 IWash Buffer I4 mL10 mLRTM1456374 JWash Buffer II2.4 mL6 mLRTM1456374 KWash Buffer III2.4 mL6 mLRTM1456374 LElution Buffer4.8 mL12 mLRT. Store in the dark.M1456374 MLoading Buffer120 µL300 µLRTM1456374 NTip pillar10T25TRTOperating Procedure: 1.Centrifuge the plasma sample (3000g, 10min), and take the supernatant for later use (if storage is required, store it at -80°C for long-term preservation to avoid repeated freezing and thawing).2.Take 50-100µL of centrifuged plasma, add 400µL of Incubation buffer I, then add 18µL of Magnetic beads, vortex to mix, and incubate at room temperature on a shaking mixer for 1 hour.3.After incubation, use a magnetic rack to magnetically separate for 3min, and discard the supernatant.4.Remove the EP tube, add 500µL of Incubation buffer II, gently invert up and down to mix several times, magnetically separate for 3min, and discard the supernatant.5.Remove the EP tube, add 500µL of Washing Buffer, gently invert up and down to mix several times, magnetically separate for 3min, and discard the supernatant.6.Remove the EP tube, add 50µL of Lysis Buffer to resuspend the beads, place in a water bath at 95°C for 10min, then take it out and cool to room temperature.7.Add 225µL of Balance buffer to the EP tube that has cooled to room temperature.8.Add 1µL of Digest buffer to the sample, and perform enzymatic digestion with shaking in a metal bath at 37°C and 1200rpm for 3-16h.9.After enzymatic digestion, take out the EP tube, add 25µL of Stop buffer to the sample, and vortex to mix.10.Add 320µL of Wash buffer I, shake vigorously for 3min, centrifuge at 15000rpm for 3min, and remove the upper liquid.11.Transfer the lower layer sample into the Tip column, centrifuge at 2500rpm for 3-5min until all the liquid is centrifuged down. If the liquid flow rate is slow, the rotation speed can be appropriately increased.12.Add 200µL of Wash buffer II (shake for 10-20s before use) to the desalting column, centrifuge at 2500rpm for 3-5min until all the liquid is centrifuged down.13.Add 200µL of Wash buffer III to the desalting column, centrifuge at 2500rpm for 3-5min until all the liquid is centrifuged down.14.Put the desalting column into a new EP tube, add 200µL of Elution buffer to the desalting column, centrifuge at 2000rpm for 3-5min until all the liquid is centrifuged down.15.Repeat the previous step, collect the eluates from both times, and freeze-dry them.16.Add 10µL of Loading buffer, vortex vigorously for 3min, centrifuge at 2000g for 10min, take an appropriate amount of sample for mass spectrometry detection. Taking the HF-X instrument as an example, 0.5-1µg of sample is sufficient for loading. Automated Operation Process:It is compatible with mass spectrometry proteomics pretreatment workstations, enabling one-stop processing from plasma to peptides. This eliminates manual operation errors, improves the reproducibility of sample preparation workflows, and provides high precision and reliability for various laboratory procedures... Read More | Live & deadtm animal cell viability / toxicity detection kit (calcein am, ethd-i) is a kit that provides double fluorescent staining for the detection of animal cell death and survival. The two probes in the kit can respectively measure the activity of cellular lactonase and the integrity of Live & deadtm animal cell viability / toxicity detection kit (calcein am, ethd-i) is a kit that provides double fluorescent staining for the detection of animal cell death and survival. The two probes in the kit can respectively measure the activity of cellular lactonase and the integrity of plasma membrane to reflect cell viability. The kit can be used for fluorescence microscopy, flow cytometry, microplate reader and other fluorescence detection systems. This kit can be applied to most eukarYOtic mammalian cells, including some tissues with adherent nuclei, but it is not applicable to fungi and yeast. Compared with trypan blue, the kit is faster, safer and more sensitive.Component： Product parameters：Calcein am: ex/em = 494 / 517 nm; Ethd-i: ex/em = 528 / 617 nm (bound DNA)Usage:Fluorescence microscopy detection1. Prepare working fluidPreparation 2 µ M Calcein AM and 4 µ M EthD-I staining solution: Remove the original solution of Calcein AM and EthD-I and restore them to room temperature. Add 20 µ L 2 mM EthD-I and 5 µ Mix 4 mM Calcein AM with 10 mL PBS or other serum-free buffer or culture medium, vortex well. The above working solution can be directly used for cell staining.Note: The aqueous solution of Calcein AM is easily hydrolyzed and should be used up every day. The concentration selection of Calcein AM and EthD-I varies depending on the type of cell used, with a recommended concentration range of 0.1-10 µ M.2. Prepare cells and conduct experiments(1) For adherent cells, they can be washed 2-3 times with 1 × PBS before staining. For suspended cells, centrifuge at room temperature of 250-1000 × g for 5 minutes and collect cells for staining.(2) Wash the cells thoroughly 2-3 times with 1 × PBS to remove residual esterase activity.(3) For adherent cells, add sufficient amount of Calcein AM/EthD-I staining solution. For suspended cells, add an appropriate amount of staining solution to control the cell density between 1-5 × 105/mL.(4) Incubate at room temperature in dark for 15-20 minutes (if the working solution concentration is high or the incubation temperature is high, the incubation time should be appropriately reduced).(5) Observe the labeled cells under a fluorescence microscope.Flow cytometry detection1. Remove the reagent and restore it to room temperature.2. Preparation 2 µ M Calcein AM and 4 µ M EthD-I staining solution: Take out the original solution of Calcein AM and EthD-I, and restore to room temperature. Add 20 µ L 2 mMEthD-I and 5 µ Vortex mix 4 mM Calcein AM with 10 mL PBS or other serum-free buffer or culture medium. The working fluid can directly stain cells.3. Wash cells thoroughly 2-3 times with 1 × PBS.4. Suspend cells with 0.5 mL of staining solution and control the cell density to 1-5 × 105/mL.Note: It is recommended to prepare two additional cell samples, each containing only one dye (Calcein AM and EthD-I), for compensatory regulation of flow cytometry single staining; Prepare another cell sample containing only buffer solution (which should be consistent with the buffer used to prepare Calcein AM and EthD-I detection working solutions) as a negative control for flow cytometry analysis.5. Incubate at room temperature in dark for 15-20 minutes.6. Within 1-2 hours, cell activity was detected by flow cytometry. Calcein AM can be excited by a 488 nm laser, with fluorescence emission spectra detected at around 530 nm and EthD-I emission spectra at around 610 nm.Note: When using the cell circle gate, attention should be paid to excluding cell debris and using a single staining tube to regulate compensation. Double staining tube flow cytometry should obtain two relatively independent cell populations: a live cell population displaying green fluorescence and a dead cell population displaying red fluorescence.ELISA reader detection1. Cultivate an appropriate amount of adherent or suspended cells in a 96 well black ELISA plate.Note: Dead cells can be obtained by treating cells with 1% saponin or 0.1-0.5% digitalis saponin for 10 minutes.2. Preparation 2 µ M Calcein AM and 4 µ M EthD-I staining solution:Remove the original solutions of Calcein AM and EthD-I and restore them to room temperature. Add 20 µ L 2 mM EthD-I and 5 µ Mix 4 mM Calcein AM 10 mL PBS or other serum-free buffer or culture medium, vortex well.Note: (1) 10 mL of staining solution is sufficient to stain a 96 well plate, and the volume of the staining solution can be adjusted according to experimental needs. The concentrations of Calcein AM and EthD-I can range from 0.1 to 10 µ Explore between M.(2) The aqueous solution of Calcein AM is easily hydrolyzed and should be used up every day. EthD-I working solution can be stored at -20 ℃ for at least one year.3. Wash the cells thoroughly with 1 × PBS to remove residual esterase activity. For adherent cells, add 100 to each well µ Wash cells with PBS. For suspended cells, add 100 µ Resuspend cells with L PBS and centrifuge to remove the supernatant. Repeat the above operation.4. Add 100 to each hole µ L PBS.5. Add 100 to each hole µ L staining solution, making the total volume of each well 200 µ L. The final concentration of Calcein AM is 1 µ M. The final concentration of EthD-I is 2 µ M. Gently shake the culture plate to evenly cover the cells with the liquid.Incubate at room temperature in dark for 30-45 minutes.Note: The optimal incubation time varies for different cells, with 30 minutes as the initial incubation time. Subsequently, the staining time can be adjusted and optimized according to the actual staining effect to obtain a more ideal staining effect.7. Enzyme reader detection. When the ELISA reader is set to fluorescein, it can detect Calcein AM; When the ELISA reader is set to rhodamine or Texas Red, EthD-I can be detected. Select the optimal emission and excitation wavelengths based on spectral characteristics.Note: By comparing the relative fluorescence values (RFU) measured between the sample group and the control group, the changes in the number of dead and live cells can be obtained. Another method of data analysis is also provided below.The following method can calculate the ratio of live cells to dead cells in a certain region. The required samples include dead cell control group, live cell control group, and the sample group to be tested. Dead cells can be obtained by treating cells with 1% saponin or 0.1-0.5% digitalis saponin for 10 minutes.1. Prepare staining solution and follow the above steps to stain cells. Additionally, prepare 1 mL and 2 mL separately µ M Calcein AM and 4 µ M EthD-I solution, stain the control group according to the following instructions. For the following groups of cells or cell-free groups, it is necessary to maintain complete consistency in cell count, detection of working solution concentration, incubation time, and incubation temperature.2. Measurement of sample group and control group:A. The measured values of the sample group at 645 nm are denoted as Calcein AM and EthD-I=F (645) sam.B. The measured values of the sample group at 530 nm are denoted as Calcein AM and EthD-I=F (530) sam.C. The measurement value of dead cell EthD-I single staining control group at 645 nm is denoted as EthD-I=F (645) maxD. The measurement value of dead cell Calcein AM single staining control group at 645 nm is recorded as Calcein AM=F (645) minE. The measurement value of live cell EthD-I single staining control group at 530 nm is recorded as EthD-I=F (530) min.F. The measurement value of live cell Calcein AM single staining control group at 530 nm is denoted as Calcein AM=F (530) max.G. A blank control well without cells (with or without dye), the detection value at 530 nm is recorded as F (530) 0.H. A blank control well without cells (with or without dye), the detection value at 645 nm is recorded as F (645) 0.3. Calculate the ratio of dead cells to live cells based on measurement data:%Live Cells=(B-E) ÷ (F-E)%Dead Cells=(A-D) ÷ (C-D)Determine the ratio of live cells to dead cells in a certain areaBy creating fluorescence spectral standard curves at 530 nm and 645 nm, the number of dead and live cells can be determined, and the fluorescence intensity of each dye is linearly related to the number of dead or live cells in the sample.Matters needing attention：1. please centrifuge the product to the bottom of the tube immediately before use, and then conduct subsequent experiments. 2. phenol red or serum may interfere with the detection of this kit. 3. fluorescent dyes have quenching problems. Please try to avoid light during experimental operation to slow down fluorescence quenching. 4. for your safety and health, please wear experimental clothes and disposable gloves.Scope of application：Dead and live cell staining (animal)... Read More | Inquire | DescriptionTakasago (R)-Ru Cymene Kit I comprises of ruthenium-based biphenyl phosphine cymene catalysts containing either BINAP and SEGPHOS®ligands. These highly reactive and selective catalysts are useful in a variety of asymmetric reactions, mainly asymmetric hydrogenation | Inquire |