Thrombospondin 1 Kit ELISA

N° du produit ABIN456570

Détail du produit Thrombospondin 1 Kit ELISA

Antigène: Thrombospondin 1 (THBS1)

Reactivité: Souris

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Gamme de detection: 7.8-500 ng/mL

Seuil minimal de détection: 7.8 ng/mL

Application: ELISA

Fonction: This immunoassay kit allows for the in vitro quantitative determination of Mouse thrombospondin 1, TSP-1 concentrations in serum, plasma and other biological fluids.

Type d'échantillon: Plasma, Serum

Analytical Method: Quantitative

Specificité: This assay recognizes recombinant and natural Mouse TSP-1.

Réactivité croisée (Details): No significant cross-reactivity or interference was observed.

Sensibilité: The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.

Attributs du produit: Mus musculus,Mouse,Thrombospondin-1,Thbs1,Tsp1

Ingrédients: 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 (1×120μl), Wash Buffer(25 x concentrate) (1×30ml), Substrate (1×10ml), Stop Solution (1×10ml), Plate sealer for 96 wells (5), Instruction (1)

Matériel non inclus: Luminometer. Pipettes and pipette tips. EP tube Deionized or distilled water.

Information d'application

Volume d'échantillon: 100 μL

Plaque: Pre-coated

Protocole: The microtiter plate provided in this kit has been pre-coated with an antibody specific to TSP-1. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for TSP-1 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 TSP-1, 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 TSP-1 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Préparation des réactifs:

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 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 500 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 (500 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml). ng/mL 500 250 125 62.5 31.2 15.6 7.8 0 Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A and B (1:100), respectively.

Prélèvement de l'échantillon: 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 -20C or -80C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 × g at 2 - 8C within 30 minutes of collection. Store samples at -20C or -80C. Avoid repeated freeze-thaw cycles. Other biological fluids - Remove particulates by centrifugation and assay immediately or aliquot and store samples at -20C or -80C. Avoid repeated freeze-thaw cycles. Note: Serum and plasma to be used within 7 days may be stored at 2-8 C, otherwise samples must stored at -20C (≤ 1 months) or -80C (≤ 2 months) to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles. When performing the assay slowly bring samples to room temperature.

Procédure de l'essai:

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37C 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 4C 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 μl of Standard, Blank, or Sample per well. Cover with the Plate sealer. Incubate for 2 hours at 37C.
2. Remove the liquid of each well, don’t wash.
3. Add 100 μl of Detection Reagent A working solution to each well. Cover with the Plate sealer. Incubate for 1 hour at 37C. Detection Reagent A working solution may appear cloudy. Warm to room temperature and mix gently until solution appears uniform. 4
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 μl) 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 μl of Detection Reagent B working solution to each well. Cover with a new Plate sealer. Incubate for 1 hours at 37C.
6. Repeat the aspiration/wash as in step
4. 7. Add 90 μl of Substrate Solution to each well. Cover with a new Plate sealer. Incubate within 30 minutes at 37C. Protect from light.
8. Add 50 μl 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 10μl 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.

Calcul des résultats:

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 TSP-1 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.

Restrictions: For Research Use only

Stockage

Conseil sur la manipulation: 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 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. 3
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.

Stock: 4 °C/-20 °C

Stockage commentaire: 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.

Détail du antigène

Antigène: Thrombospondin 1 (THBS1)

Autre désignation: Thbs1 (THBS1 Produits)

Synonymes: THBS Kit ELISA, THBS-1 Kit ELISA, TSP Kit ELISA, TSP-1 Kit ELISA, TSP1 Kit ELISA, Thbs-1 Kit ELISA, tbsp1 Kit ELISA, thrombospondin-1 Kit ELISA, thbs Kit ELISA, thbs-1 Kit ELISA, tsp Kit ELISA, tsp-1 Kit ELISA, tsp1 Kit ELISA, Tsp1 Kit ELISA, THBS1 Kit ELISA, Tsp-1 Kit ELISA, wu:fc48c03 Kit ELISA, si:dkey-11e23.1 Kit ELISA, tsp2 Kit ELISA, thrombospondin 1 Kit ELISA, thrombospondin 1 S homeolog Kit ELISA, thrombospondin 1b Kit ELISA, thrombospondin 2 L homeolog Kit ELISA, THBS1 Kit ELISA, Thbs1 Kit ELISA, thbs1.S Kit ELISA, thbs1b Kit ELISA, thbs1 Kit ELISA, thbs2.L Kit ELISA

Sujet: Thrombospondin-1 (TSP-1) is a 150 - 180 kDa charter member of the thrombospondin family of extracellular matrix proteins. The mature molecule has been described as containing three distinct regions that create the shape of a dumbbell. There is an intital, 140 aa N-terminal laminin G-like globular region that binds heparin (aa 19 - 258). This is followed by an extended, central collagen-binding region that contains one type C von Willebrand factor domain, plus three TSP type I and three TSP type II (or EGF-like) domains (aa 259 - 712). The type I repeats have multiple functions. They bind to type IV collagen, laminin, fibronectin and CD36. They also contain a recognition site for C-mannosylation on Trp. Finally, a type I KRFK motif induces the release of mature TGF-beta from LAP. This is an effect not found in TSP-2. The function of the type II repeats in unclear. The C-terminus (aa 713 - 1170) appears as a large globule with two halves, one calcium-binding region (aa 713 - 950) with seven Asp-rich TSP type III domains, and one terminal region (aa 951 - 1170) with TSP-unique motifs. This end terminal region is believed to mediate CD47 and cell binding.2 - 4 TSP-1 is secreted as a disulfide-linked 450 kDa homotrimer. The cysteines responsible lie just N-terminal to the first type I TSP repeat.

Pathways: Autophagy