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Caspase 3 ELISA 试剂盒

CASP3 适用: 人 Colorimetric Sandwich ELISA 0.312-20 ng/mL Plasma, Serum, Tissue Homogenate
产品编号 ABIN454912
发货至: 中国
  • 抗原 See all Caspase 3 (CASP3) ELISA试剂盒
    Caspase 3 (CASP3)
    适用
    • 9
    • 7
    • 6
    • 3
    • 2
    • 2
    • 2
    • 2
    • 1
    • 1
    • 1
    • 1
    检测方法
    Colorimetric
    实验类型
    Sandwich ELISA
    检测范围
    0.312-20 ng/mL
    最低检测浓度
    0.312 ng/mL
    应用范围
    ELISA
    原理
    This immunoassay kit allows for the in vitro quantitative determination of human cysteinyl aspartate specific proteinases 3, Caspase-3/CPP32 concentrations in serum, plasma , tissue homogenates and other biological fluids.
    样品类型
    Plasma, Serum, Tissue Homogenate
    Analytical Method
    Quantitative
    特异性
    This assay recognizes recombinant and natural human Caspase-3/CPP32.
    交叉反应 (详细)
    No significant cross-reactivity or interference was observed.
    灵敏度
    The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.
    产品特性
    Homo sapiens,Human,Caspase-3,CASP-3,Apopain,Cysteine protease CPP32,CPP-32,Protein Yama,SREBP cleavage activity 1,SCA-1,CASP3,CPP32,3.4.22.56
    组件
    Reagent (Quantity): Assay plate (1×20ml), Standard (2), Sample Diluent (1×20ml), Assay Diluent A (1×10ml), Assay Diluent B (1×10ml), Detection Reagent A (1×120 μl), Detection Reagent B (1×120 μl), Wash Buffer(25 x concentrate) (1×30ml), Substrate (1×10ml), Stop Solution (1×10ml), Plate sealer for 96 wells (5), Instruction (1)
    试剂未包括
    Luminometer. Pipettes and pipette tips. EP tube Deionized or distilled water.
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  • 样本量
    100 μL
    板类型
    Pre-coated
    实验流程
    2 The microtiter plate provided in this kit has been pre-coated with an antibody specific to Caspase-3/CPP32. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for Caspase-3/CPP32 and Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Then a TMB substrate solution is added to each well. Only those wells that contain Caspase-3/CPP32, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of Caspase-3/CPP32 in the samples is then determined by comparing the O.D. of the samples to the standard curve.
    试剂准备

    Bring all reagents to room temperature before use. Wash Buffer - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Dilute 30 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 750 mL of Wash Buffer. Standard - Reconstitute the Standard with 0.5 mL of Sample Diluent. This reconstitution produces a stock solution of 20 ng/ml. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions (Making serial dilution in the wells directly is not permitted). The undiluted standard serves as the high standard (20 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml). ng/mL 20 10 5 2.5 1.25 0.625 0.312 0 Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A and B (1:100), respectively. 4

    样品收集
    Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 × g. Remove serum and assay immediately or aliquot and store samples at -20 C or -80 C . Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2 - 8 C within 30 minutes of collection. Store samples at -20 C or -80 C . Avoid repeated freeze-thaw cycles. Tissue homogenates - The preparation of tissue homogenates will vary depending upon tissue type. For this assay, tissue was rinsed with 1X PBS to remove excess blood, homogenized in 20 mL of 1X PBS and stored overnight at ≤ -20 C After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged 3 for 5 minutes at 5000 x g. Remove the supernate and assay immediately or aliquot and store at ≤ -20 C Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20 C or -80 C . Avoid repeated freeze-thaw cycles. Note: Serum, plasma and tissue homogenates to be used within 7 days may be stored at 2-8 C , otherwise samples must stored at -20 C ( ≤ 1 months) or -80 C ( ≤ 2 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.
    实验流程

    Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37 C directly.). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at 4 C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their particular experiments.
    1. Add 100 μ l of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37 C .
    2. Remove the liquid of each well, don ’ t wash.
    3. Add 100 μ l of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37 C . Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform.
    4. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (approximately 400 μ l) using a squirt bottle, multi-channel pipette, manifold dispenser or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
    5. Add 100 μ l of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37 C .
    6. Repeat the aspiration/wash as in step
    4. 7. Add 90 μ l of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37 C . Protect from light.
    8. Add 50 μ l of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
    9. Determine the optical density of each well at once, using a microplate reader set to 450 nm.
    Important Note:
    1. Absorbance is a function of the incubation time. Therefore, prior to starting the assay it is recommended that all reagents should be freshly prepared prior to use and all required strip-wells are secured in the microtiter frame. This will ensure equal elapsed time for each pipetting step, without interruption.
    2. Please carefully reconstitute Standards or working Detection Reagent A and B according to the instruction, and avoid foaming and mix gently until the crystals have completely dissolved. The reconstituted Standards can be used only once. This assay requires pipetting of small volumes. To minimize imprecision caused by pipetting, ensure that pipettors are calibrated. It is recommended to suck more than 5 10 μ l for once pipetting.
    3. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. Do not allow wells to sit uncovered for extended periods between incubation steps. Once reagents have been added to the well strips, DO NOT let the strips DRY at any time during the assay.
    4. For each step in the procedure, total dispensing time for addition of reagents to the assay plate should not exceed 10 minutes.
    5. To avoid cross-contamination, change pipette tips between additions of each standard level, between sample additions, and between reagent additions. Also, use separate reservoirs for each reagent.
    6. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings.
    7. Duplication of all standards and specimens, although not required, is recommended.
    8. Substrate Solution is easily contaminated. Please protect it from light.

    结果分析

    Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit. As an alternative, construct a standard curve by plotting the mean absorbance for each standard on the x-axis against the concentration on the y-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the Caspase-3/CPP32 concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. It is recommended to use some related software to do this calculation, such as curve expert 13.0. This procedure will produce an adequate but less precise fit of the data. If samples have been diluted, the concentration read from the standard curve must be multiplied by the dilution factor.

    限制
    仅限研究用
  • 注意事项
    1. The kit should not be used beyond the expiration date on the kit label.
    2. Do not mix or substitute reagents with those from other lots or sources.
    3. If samples generate values higher than the highest standard, further dilute the samples with the Assay Diluent and repeat the assay. Any variation in standard diluent, operator, pipetting technique, washing technique,incubation time or temperature, and kit age can cause variation in binding.
    4. This assay is designed to eliminate interference by soluble receptors, ligands, binding proteins, and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.
    储存条件
    4 °C/-20 °C
    储存方法
    The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.
  • 抗原 See all Caspase 3 (CASP3) ELISA试剂盒
    Caspase 3 (CASP3)
    别名
    CASP3 (CASP3 产品)
    别名
    CPP32 ELISA Kit, CPP32B ELISA Kit, SCA-1 ELISA Kit, A830040C14Rik ELISA Kit, AC-3 ELISA Kit, Apopain ELISA Kit, CC3 ELISA Kit, Caspase-3 ELISA Kit, Lice ELISA Kit, Yama ELISA Kit, mldy ELISA Kit, xcpp32 ELISA Kit, casp3 ELISA Kit, zgc:100890 ELISA Kit, CASP-3 ELISA Kit, caspase-3 ELISA Kit, caspase 3 ELISA Kit, caspase 3 S homeolog ELISA Kit, caspase 3, apoptosis-related cysteine peptidase a ELISA Kit, caspase 3, apoptosis-related cysteine peptidase ELISA Kit, CASP3 ELISA Kit, Casp3 ELISA Kit, casp3.S ELISA Kit, casp3a ELISA Kit
    背景
    Caspases are a family of calcium-dependent cysteine proteases, which play essential roles in apoptosis (programmed cell death), necrosis and inflammation. Caspases are essential in cells for apoptosis, one of the main types of programmed cell death in development and most other stages of adult life, and have been termed ",executioner", proteins for their roles in the cell. Some caspases are also required in the immune system for the maturation of cytokines. Caspase-3, also known as CPP-32, Yama or Apopain, is an intracellular cysteine protease that exists as a proenzyme, becoming activated during the cascade of events associated with apoptosis. Caspase-3 cleaves a variety of cellular molecules that contain the amino acid motif DEVD such as poly ADP-ribose polymerase (PARP), the 70 kD protein of the U1-ribonucleoprotein and a subunit of the DNA dependent protein kinase. The presence of caspase-3 in cells of different lineages suggests that caspase-3 is a key enzyme required for the execution of apoptosis. Caspase 3 is a caspase protein. It interacts with caspase 8. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes that undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. This protein cleaves and activates caspases 6, 7, and 9, and the protein itself is processed by caspases 8, 9, and 10. It is the predominant caspase involved in the cleavage of amyloid-beta 4A precursor protein, which is associated with neuronal death in Alzheimer's disease. Alternative splicing of this gene results in two transcript variants that encode the same protein.
    途径
    Apoptosis, Caspase Cascade in Apoptosis, Sensory Perception of Sound, ER-Nucleus Signaling, Positive Regulation of Endopeptidase Activity, Activated T Cell Proliferation
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