| 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.Component100TStorageExtraction Buffer100 mL2-8℃. Store in the dark.Reagent 11EA-20℃. Store in the dark.Reagent 23EA2-8℃Reagent 31EA-20℃Reagent 415 mL2-8℃Reagent 51EA-20℃Reagent Preparation:Reagent 1 (Powder, 1 vial):Before use, centrifuge at 8000 g, 4°C for 2 min to collect the powder at the bottom (tap manually if needed).Add 1.1 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 (tap manually if needed).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 (tap manually if needed).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 (tap manually if needed).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), 96-well plate, centrifuge tubes, microplate reader, 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 microplate reader for 30 minutes. Set the wavelength to 340 nm and the temperature to 25°C.2. Thaw all reagents to room temperature (25°C).3. In a well of the 96-well plate, add sequentially:Reagent (µL)Test TubeSample20Reagent 110Reagent 210Reagent 310Reagent 4140Mix well and incubate at room temperature (25°C) for 10 minutes.4. Add Reagent (µL)Test TubeReagent 5105. 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 (T) must be substituted into the calculation formula. Alternatively, increase the sample volume appropriately (e.g., to 40 µL, with a corresponding decrease in Reagent 4 volume). The modified sample volume (V1) 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 (V1) 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 10 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 = 321.6 × 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 = 321.6 × 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 = 0.64 × ΔAParameter Description:ε: NADH molar extinction coefficient, 6.22 × 10³ L/mol/cmd: Light path of the 96-well plate, 0.5 cmV: Volume of Extraction Buffer added, 1 mLV1: Volume of sample supernatant added, 0.02 mLV2: Total reaction volume, 0.2 mL = 2.0 × 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 | The aladdin 488 Caspase-3 live cell assay kit contains the aladdin 488 Caspase-3 substrate and the Ac-DEVD-CHO Caspase-3 inhibitor. aladdin 488 Caspase-3 Substrate provides an effective tool for detecting apoptosis based on Caspase-3 activity, suitable for fluorescence microscopy and flow cytometry.The aladdin 488 Caspase-3 live cell assay kit contains the aladdin 488 Caspase-3 substrate and the Ac-DEVD-CHO Caspase-3 inhibitor. aladdin 488 Caspase-3 Substrate provides an effective tool for detecting apoptosis based on Caspase-3 activity, suitable for fluorescence microscopy and flow cytometry. Compared with other fluorescent substrates or fluorescent inhibitors of Caspase based on ( FLICA ) analysis, aladdin 488 Caspase-3 Substrate does not inhibit the apoptosis process of intact cells while detecting Caspase-3 activity. Substrate is composed of fluorescent DNA dyes coupled with Caspase-3 DEVD recognition sequence. Substrate initially had no fluorescence and entered the cytoplasm through the cell membrane. In apoptotic cells, Caspase-3 cleaves the Substrate and releases high-affinity DNA staining, which migrates to the nucleus to label DNA and emits bright green fluorescence.Therefore, aladdin 488 Caspase-3 Substrate is bifunctional, which can not only detect Caspase-3 activity, but also visualize the morphological changes of the nucleus during apoptosis. Aladdin 488 staining can be fixed in formaldehyde and compatible with subsequent immunostaining experiments.Parameters:aladdin 488：Ex/Em = 500/530 nm (with DNA)Component：Points for attention：1.Please instantaneously centrifuge the product to the bottom of the tube before use, and then carry out subsequent experiments. 2.Cells can be co-stained with a final concentration of 1µM Hoechst 33342 dye to produce blue fluorescence staining of the nucleus ( Ex / Em = 346 / 460 nm ). 3.Aladdin 488 staining can be fixed by formaldehyde, but it is not compatible with methanol fixation. 4.Formaldehyde-fixed aladdin 488-stained cells can be treated with 0.1 % TritonX-100 for subsequent staining, but the brightness of the treated staining may be weakened. 5.Fluorescent dyes all have quenching problems, please try to avoid light to slow down the fluorescence quenching. 6.For your safety and health, please wear experimental clothes and wear disposable gloves.Scope of application：Caspase 3 kit and apoptosis detectionUsage:1. Experimental optimization: The experimental steps provided below are based on the endpoint detection system. Aladdin 488 Substrate can also be used for long-term cell incubation course research. Cell density, substrate concentration, and inhibitor concentration may need to be optimized. The optimal substrate concentration may be between 1-10 µ Between M. Cells can be incubated with substrates in culture medium, PBS, or other buffer of your choice. For adherent cells, we recommend replacing them with fresh culture media containing substrates to prevent background heterogeneity. The operation of changing the medium or washing the cells after substrate incubation is freely selectable.2. We suggest that you set the following controls:A. Negative control: cells that do not induce apoptosis;B. Positive control: cells that induce apoptosis;C. Inhibitor control: Induce cell apoptosis while incubating Caspase-3/7 inhibitors (or 10-30 minutes in advance), and finally add Aladdin 488 Caspase-3 substrate.3. The Caspase-3/7 inhibitor Ac-DEVD-CHO in the Ac-DEVD-CHO Caspase-3 inhibitor control kit can be used to confirm that Caspase-3/7 depends on the fluorescence signal of aladdin 488. For inhibitor control, the final concentration of the inhibitor should be at least twice the substrate concentration (e.g. when using 5 µ At substrate M aladdin 488, the concentration of Ac-DEVD-CHO is 10 µ M). Before adding the substrate, incubate Ac-DEVD-CHO at room temperature for 15-30 minutes. After adding the substrate, continue to retain the inhibitor in the incubation solution. Ac-DEVD-CHO is a reversible competitive inhibitor. In certain cell types, effective Caspase-3/7 inhibitors require the use of irreversible inhibitors, such as Z-DEVD-FMK, or the addition of inhibitors before or during apoptosis induction.4. Flow cytometry(1) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls.(2) Adhering cells should be digested with trypsin or other methods before performing the aladdin 488 Caspase-3 experiment.(3) Resuspend cells with culture medium or buffer to achieve a cell density of 106 cells/mL(4) Suck 0.2 mL of cell suspension into a flow cytometry test tube.(5) Inhibitor control samples were treated with Ac-DEVD-CHO on cells (see 3 above) Ac-DEVD-CHO Caspase-3 inhibitor control.(6) 200 µ Add 5 to L cell suspension µ Substrate of 0.2 mM and immediately mix to achieve a substrate concentration of 5 µ M. The optimal substrate concentration for different cells may vary and requires analysis and optimization.(7) Incubate cells at room temperature in dark for 15-30 minutes.(8) Join 300 µ L-medium or PBS, analyzed by flow cytometry. Detect the channel for green fluorescence (Ex/Em=485/515 nm).5. Fluorescence microscope(1) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls.(2) Inhibitor control samples were treated with Ac-DEVD-CHO on cells (see 3 above) Ac-DEVD-CHO Caspase-3 inhibitor control.(3) Using a solution containing 5 µ M Substrate's fresh culture medium or PBS is used to replace the cell culture medium (see 1 above) Experimental optimization). For the inhibitor control group, the inhibitor was incubated together with the substrate.(4) Incubate cells at room temperature for 30 minutes or longer.(5) Cells can be directly observed in culture media containing Substrate. For the endpoint analysis method, PBS was used to clean the cells, fluorescence microscopy was used to observe the cells, and a filter (Ex/Em=485/515 nm) was used to observe green fluorescence.6. Fluorescence enzyme-linked immunosorbent assay (ELISA) reader(1) Adherent cells grow in black 96 well plates; Suspend cells, adjust the density to 106 cells/mL, and divide 0.2 mL of cell suspension into one well.(2) Choose appropriate methods to induce cell apoptosis, with untreated cell samples as controls. Note: Cells may be processed in tubes or bottles and then transferred to a 96 well detection plate.(3) Inhibitor control samples were treated with Ac-DEVD-CHO on cells (see 3 above) Ac-DEVD-CHO Caspase-3 inhibitor control.(4) For suspended cells, directly add Substrate and mix well. For adherent cells, use a solution containing 5 µ M Substrate's fresh culture medium or PBS is used to replace the cell culture medium (see 1 above) Experimental optimization). For the inhibitor control group, the inhibitor was incubated together with the substrate.(5) Cells can be directly observed in culture media containing Substrate.(6) For suspended cells, gently shake to resuspend the cells. The fluorescence enzyme-linked immunosorbent assay instrument is set with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. Suggest using bottom collection method for adherent cells. Changes in the density of adherent cells may lead to inaccurate readings... Read More | The Endo F Multi-Kit will deglycosylate N-linked glycans in both native and denatured conditions. Each enzyme has a distinct specificity for N-linked glycan release. One can choose to use the three enzymes in combination to completely remove all N-linked glycans present on a glycoprotein or peptide,The Endo F Multi-Kit will deglycosylate N-linked glycans in both native and denatured conditions. Each enzyme has a distinct specificity for N-linked glycan release. One can choose to use the three enzymes in combination to completely remove all N-linked glycans present on a glycoprotein or peptide, or to use each enzyme independently and thereby determine the type of N-glycans present.Product DescriptionThe Endo F Multi-kit is recommended to deglycosylate native proteins that are resistant to PNGase F cleavage under non-denatured conditions due to the glycan location within the protein’s three-dimensional structure, as these enzymes are known to be less sensitive to protein conformation.Each of the enzymes has a different N-linked glycan specificity:Endoglycosidase F1 cleaves high mannose and some hybrid type N-glycansEndoglycosidase F2 releases biantennary and high mannose glycans (at a 40X reduced rate)Endoglycosidase F3 will release triantennarry and fucosylated biantennary N-glycansContents1 vial: Endo F1- 20 µl (0.3 U)20 mM Tris-HCl pH 7.51 vial: Endo F2- 20 µl (0.1 U)10 mM sodium acetate, 25 mM NaCl, pH 4.51 vial: Endo F3- 20 µl (0.1 U)20 mM Tris-HCl pH 7.51 vial: 5x Reaction Buffer - 400 µl250 mM sodium acetate, pH4.51 vial: 5x Reaction Buffer - 400 µl250 mM sodium phosphate, pH5.5Specific ActivityDefined as the amount of enzyme required to catalyze the release of N-linked oligosaccharides from 1 micro-mole of denatured Ribonuclease B (Endo F1) or porcine fibrinogen peptides (Endo F2/F3) in 1 minute at 37°C, pH 5.5 (PH 4.5 for Endo F3). Cleavage is monitored by SDS-PAGE.FormulationThe enzymes are provided as a sterile-filtered solution.StabilitySeveral days exposure to ambient temperatures will not reduce activity. Stable at least 12 months when stored properly.SpecificityEndo F1 cleaves Asparagine-linked (N-linked) high mannose or hybrid oligosaccharides. Endo F2 cleaves N-linked biantennary oligosaccharides and high mannose (at a 40X reduced rate). Endo F3 cleaves free or N-linked fucosylated biantennary or triantennary oligosaccharides,as well as triamannosylchitobiose core structures. These enzymes cleave between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. The recombinant version is not glycosylated, which may result in properties differing from the native protein.Quality & PurityEndo F1, Endo F2, and Endo F3 are tested for contaminating protease as follows: 10 µg of denatured BSA is incubated at 37°C for 24 hours with 2 µl of enzyme. SDS-PAGE analysis of the treated BSA shows no evidence of degradation. The absence of exoglycosidase contaminants is confirmed by extended incubations with the corresponding pNP-glycosides. Directions for use 1. Add up to 200 µg of glycoprotein to an Eppendorf tube. Adjust to 34 µl final volume with de-ionized water. 2. Add 10 µl Endo F2 &F3 5x Reaction Buffer, 250 mM sodium acetate pH 4.5. Use Endo F1 buffer, 250 mM sodium phosphate pH 5.5 if you are using the Endo F1 enzyme alone. 4. Add 2.0 µl of each enzyme to the reaction. Incubate 3 hours at 37°C. Monitor cleavage by SDS-PAGE. Applications– Deglycosylation of native proteins resistant to PNGase F cleavage– Determination of glycan type (high mannose, biantennary, tri/tetrantennary)– Deglycosylating proteins which normally precipitate when deglycosylating– X-Ray CrystallographyThese three enzymes cleave asparagine-linked (N-linked) oligosaccharides between the two GlcNAc residues in the core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine, enhancing the solubility of the protein. In contrast, PNGase F removes the oligosaccharide intact... Read More | The content of this cell is too long for an XLSX file (more than 32767 characters). Please use the CSV format for this export | Inquire |