Calretinin ELISA Kit

Produktnummer ABIN454633

Produktdetails Calretinin ELISA Kit

Target: Calretinin (CALB2) (Calbindin 2 (CALB2))

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Detektionsbereich: 78-5000 pg/mL

Untere Nachweisgrenze: 78 pg/mL

Applikation: ELISA

Verwendungszweck: This immunoassay kit allows for the specific measurement of human Calretinin concentrations in cell culture supernates, serum, and plasma.

Proben: Cell Culture Supernatant, Serum, Plasma

Analytische Methode: Quantitative

Spezifität: This assay recognizes recombinant and natural human Calretinin.

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

Produktmerkmale: Homo sapiens,Human,Calretinin,CR,29 kDa calbindin,CALB2,CAB29

Bestandteile: Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A 1×120µl Detection Reagent B 1×120µl Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)

Anwendungsinformationen

Probenmenge: 100 μL

Plattentyp: Pre-coated

Protokoll: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Calretinin has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Calretinin present is bound by the immobilized antibody. An enzyme-linked polyclonal antibody specific for Calretinin is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of Calretinin bound in the initial step. The color 2 development is stopped and the intensity of the color is measured.

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 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 3 produces a stock solution of 50 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making serial dilutions. The undiluted standard serves as the high standard (50 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.

Probennahme: 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.

Testdurchführung:

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.
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
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.

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 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 Calretinin 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.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

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 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.

Details zu Calretinin

Target: Calretinin (CALB2) (Calbindin 2 (CALB2))

Andere Bezeichnung: CALB2 (CALB2 Produkte)

Synonyme: CAB29 ELISA Kit, CAL2 ELISA Kit, CR ELISA Kit, calb2l ELISA Kit, wu:fq18e08 ELISA Kit, zgc:73115 ELISA Kit, calb2 ELISA Kit, wu:fq17g09 ELISA Kit, zgc:73099 ELISA Kit, calbindin 2 ELISA Kit, calbindin 2a ELISA Kit, calbindin 2b ELISA Kit, CALB2 ELISA Kit, Calb2 ELISA Kit, calb2a ELISA Kit, calb2b ELISA Kit

Hintergrund: Calretinin is a calcium-binding protein, 29 kDa, member of the family of so-called EF-hand proteins, to which also the S-100 proteins belong. Calretinin is abundantly expressed in neurons. Outside the nervous system, calretinin is found in mesothelial cells, steroid producing cells (adrenal cortical cells, testicular Leydig cells, ovarian theca interna cells), testicular Sertoli cells, rete testis, ovarian surface epithelium, some neuroendocrine cells, breast glands, eccrine sweat glands, hair follicular cells, thymic epithelial cells, endometrial stromal cells, and fat cells. In calretinin positive cells, the protein is generally found in both the cytoplasm and nuclei. Calretinin is also considered an important diagnostic aid in the differential diagnosis of cystic and solid ameloblastic tumors. The rat and human calretinin exhibit 98% sequence homology and 91% homology to many other species. Calretinin is generally expressed with two other calcium binding proteins Calbindin D-29 and parvalbumin in CNS and other peripheral tissues. Two calcium binding proteins, calbindin and calretinin, have been reported to be expressed in abundance in Purkinje cells and other cell types in the cerebellum. Calretinin is an intracellular calcium-binding protein belonging to the troponin C superfamily characterised by a structural motif described as the EF-hand domain. Calcium is an important moderator of a number of vital physiological processes, including neuronal excitability, axonal transport, synthesis and release of some neurotransmitters, membrane permeability and enzyme activity. Calretinin possesses six characteristic EF-hand structures of which five are able to bind calcium ions with high affinity. Calretinin is found in the nervous system and thymus. Calretinin can also be demonstrated at precise moments of the cell cycle, in particular, G1 phase and mitosis, and is associated with kinetochore microtubules which may indicate a role in chromosome separation.