| Description | Background information Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its Background information Annexins are a family of calcium-dependent phospholipid-binding proteins that preferentially bind phosphatidylserine (PS). Under normal physiologic conditions, PS is predominantly located in the inner leaflet of the plasma membrane. Upon initiation of apoptosis, PS loses its asymmetric distribution across the phospholipid bilayer and is translocated to the extracellular membrane leaflet marking cells as targets of phagocytosis. Once on the outer surface of the membrane, PS can be detected by fluorescently labeled Annexin V in a calcium-dependent manner.In early-stage apoptosis, the plasma membrane excludes viability dyes such as propidium iodide (PI), 7-AAD. These cells will stain with Annexin V but not a viability dye, thus distinguishing cells in early apoptosis. However, in late stage apoptosis, the cell membrane loses integrity thereby allowing Annexin V to also access PS in the interior of the cell. A viability dye can be used to resolve these late-stage apoptotic and necrotic cells (Annexin V, viability dye-positive) from the early-stage apoptotic cells (Annexin V positive, viability dye-negative).We offer recombinant Annexin V conjugated to a numerous fluorophores, as well as an Annexin V biotin conjugate which can be detected with fluorophore-labeled streptavidin. By binding to PS, fluorophores labeled Annexin V can be used to detect and quantify apoptotic cells via flow cytometry or fluorescence microscopy. The excitation and emission maxima of the Annexin V conjugates are summarized in the following table.Cat.No.Ex/Em (nm)Formatrp226056NABiotinrp225999401/422AF405rp226057490/525AF488rp226060650/668AF647rp226002681/704AF680rp226003752/776AF750rp226053498/517FITCrp226004410/455Pacific Bluerp226006647/665Cy5rp226054650/660APCrp226055565/575PErp226058565/670PE-Cy5rp226059565/774PE-Cy7Precautions1. Please try to avoid light when using to slow down the quenching of fluorescence.2. Propidium Iodide Solution is toxigenic and mutagenic; handle with care.3. Due to the calcium dependence of the Annexin V:PS interaction, it is critical to avoid buffers containing EDTA or other calcium chelators during Annexin V experiments.Instruction for use1. Dilute 10X Binding Buffer (A1372288) to 1X using distilled water (1 mL 10X Binding Buffer + 9 mL ddH2O).2. Wash cells twice with cold PBS and then resuspend the desired amounts of cells in Annexin V Binding Buffer at a concentration of 1.0-5.0 x 106 cells/mL.3. Add 5 µL of Annexin V-AF405 to 100 µL of the cell suspension. Stain with a viability dye, such as PI (P1373641; P1372285), 7-AAD (A1372406), or DAPI (D1372407) dyes, if desired.4. Gently vortex the cells and incubate for 10 min at RT (25°C) in the dark.5. Add 100 µL of 1X Binding Buffer to each assay. Analyze by flow cytometry within 1 hr... Read More | Inquire | Acid phosphatase is an esterase with broad activity at an optimal pH below 7.0. There are three isozymes, EI, EII, and EIII of similar molecular weight (55 kDa± 5 kDa). Their optimum pH's are 5.5, 4.5, and 4.0 respectively. Acid phosphatase activity was observed by Teller Aladdin Library Acid phosphatase is an esterase with broad activity at an optimal pH below 7.0. There are three isozymes, EI, EII, and EIII of similar molecular weight (55 kDa± 5 kDa). Their optimum pH's are 5.5, 4.5, and 4.0 respectively. Acid phosphatase activity was observed by Teller Aladdin Library Archives in 1954 in preparations of a wheat germ lipase described by Singer JBC, 174, 11, in 1948. Equivalent commercial preparations have been distributed labeled as lipase and acid phosphatase thus generating some confusion. Subsequent work has confirmed that the non-specific esterase activity of the wheat germ preparation may be measured both as lipase (triacetin as substrate) and phosphatase. The enzyme assay is based on the work of Brandenberger and Hanson (Helv. Chim. Acta, 36, 900, 1953) and Hofstee ( Arch. Biochem. Biophys., 51, 239, 1954).Acid phosphatase (APase) non-specifically catalyzes the hydrolysis of monoesters and anhydrides of phosphoric acid to produce inorganic phosphate. It is used to study the production, transport, and recycling of phosphate and the metabolic and energy transduction processes of the cell.Characteristics of Acid Phosphatase from Wheat Germ:Molecular weight: 55,000 ± 5,000 (Verjee 1969).Composition: Three isozymes of closely similar molecular weights have been reported by Verjee (1969): EI, EII, and EIII. See also Brouillard and Ouellet (1965).Optimal pH: EI - 5.5, EII - 4.5, and EIII - 4.0. (Verjee 1969).Specificity: The enzyme has a broad esterase activity. See Joyce and Grisolia (1960). It shows highest activity for pyrophosphate.Inhibitors: Fluoride, molybdate and orthophosphate (Verjee 1969)... Read More | Inquire | TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for measuring HRP probe activity. A stable blue precipitate is formed at the reaction site.The substrate does not contain NMP (1-methyl2-pyrrolidone) making it REACH Restricted Substances List Annex XVII compliant, while ensuring maximal safety during use, and minimal negative environmental impact.Product Characteristics TMB (3, 3', 5, 5'-tetramethylbenzidine) is a chromogenic substrate for Horseradish Peroxidase (HRP). TMB produces a deep blue color during the enzymatic degradation of hydrogen peroxide by HRP.TMB-D Blotting liquid ready-to-use substrate is a highly active and stable blotting substrate utilized for measuring HRP probe activity. A stable blue precipitate is formed at the reaction site. The substrate does not contain NMP (1-methyl-2- pyrrolidone) making it REACH Restricted Substances List Annex XVII compliant, while ensuring maximal safety during use, and minimal waste problems after use.Composition & Properties Ready-to-use substrate: Includes substrate buffer and hydrogen peroxide. No other reagents should be added.Working Procedure The following procedure is applicable to nitrocellulose membranes. The procedure must be optimized for other membranes.1.The desired amount of substrate is poured into a sealed container and allowed to reach room temperature, in the dark, before use. 2.After the last incubation with HRP-labelled Streptavidin or HRP-labelled secondary antibody it is recommended to wash the membrane in a 0.1 M Tris buffer pH 7.4.3.Shake off the excess buffer and incubate the membrane in the TMB-D Blotting solution for 10 minutes. 4.Wash the membrane in distilled water and allow it to dry. 5.The site of positive reaction will appear light blue with no or very little background staining.Tips & Tricks • The membrane can be blocked with Kementec’s Synthetic Blocking Buffer for Blotting, (cat. no. S494457). • For long-term preservation of the results, the membranes must be stored in the dark.Handling & Storage • Store solution at 2-8⁰C in the dark. • Avoid exposure to light, heat and contamination with metal ions or peroxidase. • Re-dispense only into bottles made of High-Density Polyethylene (HDPE), amber color. Dispensing guidelines are available upon request... Read More |