Slit Homolog 2 (Drosophila) (SLIT2) ELISA Kit

Antigen: Slit Homolog 2 (Drosophila) (SLIT2)

Synonyme: SLIL3, Slit-2, FLJ14420, Slil3, Drad-1, KIAA4141, mKIAA4141, E030015M03Rik, E130320P19Rik, Slit, slit2-a

Reaktivität: Human

Applikation: ELISA

Produktnummer: ABIN578927

Produktbeschreibung

Produktmerkmale: This immunoassay kit allows for the in vitro quantitative determination of human Slit2 concentrations in cell culture supernates, serum, plasma and other biological fluids.

Weitere Bezeichnung: Slit2

Gen-ID: 3100

Beschreibung: Slit2 plays a vital role in axon guidance by signaling through Robo receptors. Recent evidence suggests that Slit2 protein may function in other settings because human and Xenopus Slit2 has been shown to inhibit leukocyte chemotaxis. SLIT2 protein is a putative ligand for the ROBO receptors. ROBO1 is inactivated by promoter region hypermethylation in <,20% of human cancers. SLIT2 is an excellent candidate tumor suppressor gene for colorectal cancer. SLIT2 has tumor suppressor activity and that it is epigenetically silenced in >,40% of lung and breast tumors. SLIT2 promoter region methylation was found in 23 (72%) of 32 primary colorectal cancers. In contrast, normal colorectal mucosa from the same patients exhibited significantly lower levels of SLIT2 promoter region hypermethylation. SLIT2 methylation was reversed and expression restored by treating colorectal tumor cell lines with the demethylating agent 5-aza-2-deoxycytidine. Loss of heterozygosity at D4S1546 marker, which maps within 100 kb of the SLIT2 gene, was observed in 39% of the methylated tumors. Furthermore, SLIT2 epigenetic silencing was independent of ROBO1/p16/RASSF1A hypermethylation. The presence of SLIT2 methylation was also independent of the presence of K-RAS mutations. Ectopic expression of SLIT2 diminished the ability to form colonies in two colorectal tumor cell lines. In addition, conditioned medium from SLIT2-transfected COS-7 cells reduced cell growth and induced apoptosis in SW48 colorectal tumor cell line.

Spezifität: This assay recognizes recombinant and natural human Slit2. No significant cross-reactivity or interference was observed.

Sensitivität: The minimum detectable dose of human Slit2 is typically less than 0.078 ng/ml. The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest protein concentration that could be differentiated from zero. 0.156-10 ng/mL. The standard curve concentrations used for the ELISA ’ s were 10 ng/mL, 5 ng/mL, 2.5 ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.312 ng/mL, 0.156 ng/mL.

Anwendungen

Prinzip: The microtiter plate provided in this kit has been pre-coated with an antibody specific to Slit2. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for Slit2 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 Slit2, 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 2 nm. The concentration of Slit2 in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Protokoll: Sample collection and storage:
Cell culture supernates - Remove particulates by centrifugation and assay immediately or aliquot and store samples at ≤ -20° C. Avoid repeated freeze-thaw cycles. Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes before centrifugation for 15 minutes at approximately 1000 x g. Remove serum and assay immediately or aliquot and store samples at -20° C. Plasma - Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000 x g at 2 - 8° C within 30 minutes of collection. Store samples at ≤ -20° C. Avoid repeated freeze-thaw cycles. Note: Citrate plasma has not been validated for use in this assay.
Limitations of the procedure:
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.
Reagent preparation:
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 20 mL of Wash Buffer Concentrate into deionized or distilled water to prepare 500 ml of Wash Buffer. Standard - Reconstitute the Standard with 1.0 mL of Sample Diluent. This reconstitution produces a stock solution of 800 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The diluted standard serves as the high standard (400 ng/mL). The Sample Diluent serves as the zero standard (0 ng/mL). Detection Reagent A and B - Dilute to the working concentration specified on the vial label using Assay Diluent A and B (1:100), respectively.
Assay procedure:
Allow all reagents to reach room temperature. Arrange and label required number of strips. 1. Prepare all reagents, working standards and samples as directed in the previous sections. 2. Add 100 uL of Standard, Control, or sample* per well. Cover with the adhesive strip. Incubate for 2 hours at 37° C. 3. Remove the liquid of each well, don’t wash. 4. Add 100 uL of Detection Reagent A to each well. Incubate for 1 hour at 37°C. Detection Reagent A may appear cloudy. Warm to room temperature and mix gently until solution appears uniform. 3 5. Aspirate each well and wash, repeating the process three times for a total of three washes. Wash by filling each well with Wash Buffer (350 uL) 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. 6. Add 100 uL of Detection Reagent B to each well. Cover with a new adhesive strip.Incubate for 1 hours at 37° C. 7. Repeat the aspiration/wash as in step 5. 8. Add 90 uL of Substrate Solution to each well. Incubate for 30 minutes at room temperature. Protect from light. 9. Add 50 uL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. 10. Determine the optical density of each well within 30 minutes, using a microplate reader set to 450 nm.
Important Note:
1. The wash procedure is critical. Insufficient washing will result in poor precision and falsely elevated absorbance readings. 2. It is recommended that no more than 32 wells be used for each assay run if manual pipetting is used since pipetting of all standards, specimens and controls should be completed within 5 minutes. A full plate of 96 wells may be used if automated pipetting is available. 3. Duplication of all standards and specimens, although not required, is recommended. 4. When mixing or reconstituting protein solutions, always avoid foaming. 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. To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary. 4. Calculation of results: 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 y-axis against the concentration on the x-axis and draw a best fit curve through the points on the graph. The data may be linearized by plotting the log of the ADR concentrations versus the log of the O.D. and the best fit line can be determined by regression analysis. 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.

Bestandteile: Reagent (Quantity ): Assay plate (1×20ml), Standard (2), Sample Diluent (1 × 20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A (1 × 120μl), Detection Reagent B (1 × 120μl), Wash Buffer (25 x concentrate) (1 × 30ml), Substrate (1x10ml), Stop Solution (1x10ml), Plate sealer for 96 wells (5), Instruction (1)

Benötigtes Material: Luminometer. Pipettes and pipette tips. EP tube Deionized or distilled water.

Lagerung: 1. Unopened test kits should be stored referring to the package label for frequent use, and stored at -20 C for long time storage. The unused strips should be kept in a sealed bag and stored at 2-8 C in their pouch with the desiccant provided to minimize exposure to damp air. The test kit may be used throughout the expiration date of the kit (six months from the date of manufacture). Opened test kits will remain stable until the expiring date shown, provided it is stored as prescribed above. 2. There may be some foggy substance in the wells when the plate is opened at the first time. It will not have any effect on the final assay results. 3. Do not remove microtiter plate from the storage bag until needed. 4. A microtiter plate reader with a bandwidth of 10nm or less and an optical density range of 0-3 OD or greater at 450nm wavelength is acceptable for use in absorbance measurement. 5. Use fresh disposable pipette tips for each transfer to avoid contamination. 6. Do not substitute reagents from one kit lot to another. Use only the reagents supplied by manufacturer. 7. Valid period: six months. 6.

Beschränkungen: Nur für Forschungszwecke einsetzbar