| Description | 3-Phosphoglycerate kinase (PGK) is a key enzyme in glycolysis, widely present in animals, plants, and microorganisms. It catalyzes the reaction of 3-phosphoglycerate and ATP to produce 1,3-bisphosphoglycerate. The latter, under the action of glyceraldehyde-3-phosphate dehydrogenase and NADH, 3-Phosphoglycerate kinase (PGK) is a key enzyme in glycolysis, widely present in animals, plants, and microorganisms. It catalyzes the reaction of 3-phosphoglycerate and ATP to produce 1,3-bisphosphoglycerate. The latter, under the action of glyceraldehyde-3-phosphate dehydrogenase and NADH, produces glyceraldehyde-3-phosphate and NAD+. The activity of 3-phosphoglycerate kinase (PGK) is determined by measuring the decrease in NADH.Component50TStorageExtraction Buffer50 mL2-8℃. Store in the dark.Reagent 11EA-20℃. Store in the dark.Reagent 23EA2-8℃Reagent 31EA-20℃Reagent 435 mL2-8℃Reagent 51EA-20℃Reagent Preparation:Reagent 1 (Powder, 1 vial):Before opening, ensure the powder is at the bottom of the vial (tap manually if needed).Add 2.2 mL of distilled water to dissolve. The dissolved reagent can be aliquoted and stored at -20°C.Reagent 2 (Powder, 3 vials):Before use, centrifuge at 8000 g, 4°C for 2 min to collect the powder at the bottom.Add 0.4 mL of distilled water to dissolve. The dissolved reagent can be aliquoted and stored at -20°C (use within one month after dissolution).Reagent 3 (Liquid, 1 vial):Before use, centrifuge at 8000 g, 4°C for 2 min to collect the liquid at the bottom.Add 1.1 mL of distilled water to dissolve. The dissolved reagent can be aliquoted and stored at -20°C.Reagent 5 (Powder, 1 vial):Before use, centrifuge at 8000 g, 4°C for 2 min to collect the powder at the bottom.Add 1.1 mL of distilled water to dissolve.The storage period is the same as the kit's expiry date.User-Prepared Instruments & MaterialsMortar (homogenizer), ice bucket (ice maker), benchtop centrifuge, adjustable pipettes, water bath (oven, incubator, metal bath), 1 ml quartz cuvette, centrifuge tubes, UV spectrophotometer, distilled water (deionized water or ultrapure water is acceptable).Sample Extraction1. Tissue Samples: Weigh approximately 0.1 g of tissue, add 1 mL of Extraction Buffer, homogenize on ice, and then centrifuge at 12000 rpm, 4°C for 5 minutes. Collect the supernatant for assay.Note: If increasing the sample amount, use a ratio of 1:5 to 1:10 (tissue weight (g) : Extraction Buffer volume (mL)) for extraction.2. Bacterial/Cell Samples: Collect bacteria or cells into a centrifuge tube by centrifugation and discard the supernatant. Take approximately 5 million bacteria or cells, add 1 mL of Extraction Buffer, and disrupt using ultrasound on ice (power 200 W, ultrasonicate for 3 s, interval 10 s, repeat 30 times). Centrifuge at 12000 rpm, 4°C for 10 minutes. Collect the supernatant and keep it on ice for assay.Note: If increasing the sample amount, use a ratio of 500-1000 (x10⁴ cells) : 1 (mL Extraction Buffer) for extraction.Assay Procedure1. Preheat the UV spectrophotometer for 30 minutes. Set the wavelength to 340 nm and the temperature to 25°C. Zero the instrument with distilled water.2. Thaw all reagents to room temperature (25°C).3. In a 1 mL quartz cuvette (1 cm light path), add sequentially:Reagent (µL)Test TubeSample80Reagent 140Reagent 220Reagent 320Reagent 4600Mix well and incubate at room temperature (25°C) for 10 minutes.4. Add Reagent (µL)Test TubeReagent 5205. Mix gently. At room temperature (25°C), read the absorbance at 340 nm at 30 seconds (A1) and then again after 10 minutes (A2). Calculate ΔA = A1 - A2.Notes:1. If ΔA is close to zero, the reaction time can be appropriately extended to 20 minutes before reading A2. The modified reaction time must be substituted into the calculation formula. Alternatively, increase the sample volume appropriately (e.g., 100 µL, with a corresponding decrease in Reagent 4 volume). The modified sample volume must be substituted into the calculation formula.2. If the decreasing trend is unstable, read the absorbance every 20 seconds and select a linear decreasing period for calculation. The corresponding ΔA value should be substituted into the calculation formula.3. If the initial absorbance A1 is too high (e.g., >2, as in deeply pigmented plant leaves), appropriately reduce the sample volume. The modified sample volume must be substituted into the calculation formula. Alternatively, add a small amount of activated carbon to the sample, mix, let stand for 5 min, then centrifuge at 12000 rpm, 4°C for 10 min, and use the supernatant for detection.4. If ΔA is greater than 0.5, reduce the reaction time (e.g., to 5 min) or reduce the sample volume (e.g., to 20 µL). The modified reaction time (T) and sample volume (V1) must be substituted into the calculation formula.PGK Activity Calculation1. Based on Sample Mass:Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per gram of tissue.Formula:PGK (nmol/min/g fresh weight) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (W × V1 ÷ V) ÷ T = 156.8 × ΔA ÷ W2. Based on Sample Protein Concentration:Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per mg of protein.Formula:PGK (nmol/min/mg prot) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (V1 × Cpr) ÷ T = 156.8 × ΔA ÷ Cpr3. Based on Bacterial/Cell Count:Unit Definition: One unit of enzyme activity is defined as the consumption of 1 nmol NADH per minute per 10⁴ cells.Formula:PGK (nmol/min/10⁴ cell) = [ΔA ÷ (ε × d) × V2 × 10⁹] ÷ (500 × V1 ÷ V) ÷ T = 0.314 × ΔAParameter Description:ε: NADH molar extinction coefficient, 6.22 × 10³ L/mol/cmd: Cuvette light path, 1 cmV: Volume of Extraction Buffer added, 1 mLV1: Volume of sample supernatant added, 0.08 mLV2: Total reaction volume, 0.78 mL = 7.8 × 10⁻⁴ LT: Reaction time, 10 minW: Sample mass, g500: Cell number, in units of 10⁴Cpr: Protein concentration of the supernatant, mg/mL; Aladdin BCA Protein Quantification Kit (B665595) or Ready-to-Use BCA Protein Quantification Kit (R1491648) are recommended.PrecautionsIt is recommended to first select 1-3 samples with significant differences (e.g., different types or groups) for preliminary experiments to familiarize yourself with the procedure. Determine or adjust the sample concentration based on the preliminary results to prevent unnecessary waste of samples or reagents... Read More | Product Characteristics Effect Diluents, Animal-free are effective buffers free of any animal components. They can be used for the dilution of serum, plasma, blood, stool or urine samples, as well as the dilution of primary and secondary antibodies. Effect Diluents, Animal-free efficiently minimize Product Characteristics Effect Diluents, Animal-free are effective buffers free of any animal components. They can be used for the dilution of serum, plasma, blood, stool or urine samples, as well as the dilution of primary and secondary antibodies. Effect Diluents, Animal-free efficiently minimize matrix effects, cross-reactions and unspecific binding in immunoassays like ELISA, Western blotting, Immunohistochemistry, protein arrays and immuno-PCR.The Effect Diluents, Animal-free are used alternatively to the standard sample or antibody dilution buffers: In ELISA for the dilution of specimen and detection antibodies. In Western Blotting for the dilution of primary and secondary antibodies. In Protein arrays for the dilution of specimen and detection antibodies. In immuno-PCR as a washing buffer.Three versions of the diluent are offered: Low, Medium and High for optimal discrimination between specific and unspecific reaction and for minimizing strong interference effects e.g., by RF (rheumatoid factors), HAMAs (human-a-mouse Abs) or by endogenous components that bind and mask the analyte.Composition & Properties The Effect Diluents, Animal free contain no animal components and are free of phosphates.Working Procedure 1.Mix thoroughly prior to use. 2.Dilution recommendations a.Dilute antibodies according to the instruction of the antibody b.Dilution of the specimen is recommended at 1:2 or higherTips & TricksEffect Diluents must not be considered as blocking buffers. Recommended blocking buffers are: Synthetic Blocking Buffer, ELISA (cat. no. S494401), Synthetic Blocking Buffer, Blotting (cat. no. S494457) and WellChampion (cat. no. W494467) for plate blocking and stabilization (preparation of pre-coated plates). Complex sample matrices, such as serum and plasma, may contain interfering factors that affect the ability of the assay to accurately quantify the target analyte. Strong interferences are often caused by RFs and HAMAs. This matrix effect can cause high background in the negative control or false negatives in the sample measurement. To reduce this effect the samples can be diluted in the Effect Diluents, Animalfree.Handling & Storage Store solution 2-8°C or -15 to -30°C (tolerates freezing and thawing cycles)... Read More | Products Content:F666101Component500 U5000 UStorageF666101AFastStar Probe Buffer (for bisDNA)2×1.2 mL2×12 mL-20℃. Avoid freeze/thaw cycle. Protect from light.F666101BSuperFastStar DNA Polymerase (5U/µL)100 µL1 mL-20℃. Avoid freeze/thaw cycle. Protect from light.Products Content:F666101Component500 U5000 UStorageF666101AFastStar Probe Buffer (for bisDNA)2×1.2 mL2×12 mL-20℃. Avoid freeze/thaw cycle. Protect from light.F666101BSuperFastStar DNA Polymerase (5U/µL)100 µL1 mL-20℃. Avoid freeze/thaw cycle. Protect from light.Products IntroductionThis product is mainly used for PCR using bisulfite-treated DNA as template, in which SuperFastStar DNA Polymerase is a new high-efficiency hot-start enzyme modified by bis-monoclonal antibody, which is completely blocked at room temperature, thus effectively avoiding non-specific amplification caused by the non-specific binding of the primer to the template or the primer dimerization under the condition of room temperature. The optimized FastStar Probe Buffer (for bisDNA) contains PCR Buffer, dNTPs and Mg2+, etc., which is easy to use as customers only need to add templates, primers and probes.caveat1 Before use, please mix the product gently by turning it up and down after it has been completely melted and centrifuged briefly.2. Avoid repeated freezing and thawing of the product, which may degrade its performance. This product can be stored at -20℃ for a long period of time, protected from light. If frequent use is required within a short period of time, it can be stored at 2-8℃.Usage The following examples are conventional PCR reaction systems and conditions, which should be improved and optimized according to the template, primer structure and target fragment size.1.PCR reaction system Note: 1) Usually, better results can be obtained with a primer concentration of 0.2 µM, and 0.1-1.0 µM can be used as a reference for setting the range.2）The concentration of the probe used is related to the fluorescence quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the instrument manual or the specific requirements for the use of each fluorescent probe to adjust the concentration.3）Usually the amount of DNA template is 10-100 ng of genomic DNA or 1-10 ng of cDNA as a reference. Since the templates of different species contain different copy numbers of the target gene, the templates can be diluted in gradients to determine the optimal amount of template to use.2.PCR reaction conditionsNote: 1) The initial denaturation of this product at 95°C for 30s is sufficient for enzyme activation; complex templates can be extended to 3min denaturation.(2) It is recommended to use two-step PCR reaction program, if you can't get good experimental results due to the use of primers with lower Tm value, etc., you can try three-step PCR amplification, and the annealing temperature should be set in the range of 56℃-64℃ as a reference... 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 | 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 |