Gelsolin ELISA Kit (GSN)

Details for Product GSN ELISA Kit No. ABIN578803, Anbieter: Anmelden zum Anzeigen
Antigen
  • CG1106
  • DGS
  • Dmel\\CG1106
  • gel
  • scin
  • ADF
  • AGEL
  • cb499
  • fb94b12
  • fc04g09
  • gsn
  • gsnl1
  • sb:cb499
  • wu:fc04g09
  • gelsolin
  • Gelsolin
  • gelsolin S homeolog
  • scinderin like a
  • GSN
  • Gel
  • gsn
  • Gsn
  • gsn.S
  • scinla
Reaktivität
Ratte (Rattus)
Alternativen
Kits mit alternativen Reaktivitäten:
21
13
8
8
5
4
4
3
2
1
1
1
1
Methodentyp
Sandwich ELISA
Detektionsbereich
6.25-400 ng/mL
Untere Nachweisgrenze
6.25 ng/mL
Applikation
ELISA
Optionen
Hersteller
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Hersteller Produkt- Nr.
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Verwendungszweck This immunoassay kit allows for the in vitro quantitative determination of rat Gelsolin concentrations in cell culture supernates, serum, plasma and other biological fluids.
Proben Cell Culture Supernatant, Plasma, Serum
Analytische Methode Quantitative
Nachweismethode Colorimetric
Spezifität This assay recognizes recombinant and natural rat Gelsolin.
Kreuzreaktivität (Details) No significant cross-reactivity or interference was observed.
Sensitivität The sensitivity of this assay, or Lower Limit of Detection (LLD) was defined as the lowest detectable concentration that could be differentiated from zero.
Produktmerkmale Rattus norvegicus,Rat,Gelsolin,Actin-depolymerizing factor,ADF,Brevin,Gsn
Bestandteile Reagent (Quantity ): Assay plate (1×20ml), Standard (2), 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), Instructions (1)
Benötigtes Material Luminometer. Pipettes and pipette tips. EP tube Deionized or distilled water.
Andere Bezeichnung Gsn (GSN ELISA Kit Abstract)
Hintergrund Gelsolin is an actin-regulatory protein that modulates actin assembly and disassembly, and is believed to regulate cell motility in vivo through modulation of the actin network. In addition to its actin-regulatory function, gelsolin has also been proposed to affect cell growth. It is first isolated from rabbit lung macrophages as a modulator of the cytoplasmic actin gel−sol transformation. Gelsolin can sever actin filaments and cap the fast-growing ends of the filaments in vitro, this promotes actin disassembly. Gelsolin also has the ability to nucleate actin polymerization. These functions are activated by Ca2+ and inhibited by polyphosphoinositides (PPIs). Such observations have led to the view that gelsolin regulates actin reorganization in response to changes in the concentrations of Ca2+ and PPIs in living cells.
Gen-ID 3183
Pathways Caspase Kaskade in der Apoptose, Regulation of Actin Filament Polymerization, Autophagie
Probenmenge 100 μL
Plattentyp Pre-coated
Protokoll The microtiter plate provided in this kit has been pre-coated with an antibody specific to Gelsolin. Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated polyclonal antibody preparation specific for Gelsolin 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 Gelsolin, 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 Gelsolin in the samples is then determined by comparing the O.D. of the samples to the standard curve.
Aufbereitung der Reagenzien

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 200 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 (200 ng/ml). The Sample Diluent serves as the zero standard (0 ng/ml). ng/mL 200 100 50 25 12.5 6.25 3.12 0 Detection Reagent A and B - Dilute to the working concentration using Assay Diluent A and B (1:100), respectively.

Probennahme 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 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 -20C or -80C. Avoid repeated freeze-thaw cycles. Note: Tissue homogenates 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.
Testdurchführung

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 37°C. Detection Reagent A working solution may 4 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 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 37°C. 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.

Ergebnisberechnung

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 MYS 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.

Beschränkungen Nur für Forschungszwecke einsetzbar
Handhabung 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 3 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.
Lagerung 4 °C/-20 °C
Informationen zur Lagerung 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.
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