GRP94 Kit ELISA

N° du produit ABIN455313

Détail du produit GRP94 Kit ELISA

Antigène: GRP94 (HSP90B1) (Heat Shock Protein 90kDa beta (Grp94), Member 1 (HSP90B1))

Reactivité: Humain

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Gamme de detection: 0.156-10 ng/mL

Seuil minimal de détection: 0.156 ng/mL

Application: ELISA

Fonction: This immunoassay kit allows for the specific measurement of Human Glycoprotein 96 concentrations in cell culture supernates, serum, and plasma.

Type d'échantillon: Cell Culture Supernatant, Serum, Plasma

Analytical Method: Quantitative

Specificité: This assay recognizes recombinant and natural Human Glycoprotein 96.

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

Attributs du produit: Homo sapiens,Human,Endoplasmin,94 kDa glucose-regulated protein,GRP-94,Heat shock protein 90 kDa beta member 1,Tumor rejection antigen 1,gp96 homolog,HSP90B1,GRP94,TRA1

Ingrédients: Reagent (Quantity): Assay plate (1), 2 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)

Information d'application

Volume d'échantillon: 100 μL

Plaque: Pre-coated

Protocole: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for Glycoprotein 96 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any Glycoprotein 96 present is bound by the immobilized antibody. An enzyme-linked monoclonal antibody specific for Glycoprotein 96 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 Glycoprotein 96 bound in the initial step. The color development is stopped and the intensity of the color is measured.

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 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 10 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 (10ng/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 3 Assay Diluent A and B (1:100), respectively.

Prélèvement de l'échantillon: 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.

Procédure de l'essai:

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. 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.

Calcul des résultats:

4 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 Glycoprotein 96 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.

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 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.

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: GRP94 (HSP90B1) (Heat Shock Protein 90kDa beta (Grp94), Member 1 (HSP90B1))

Autre désignation: HSP90B1 (HSP90B1 Produits)

Synonymes: ecgp Kit ELISA, gp96 Kit ELISA, tra1 Kit ELISA, grp94 Kit ELISA, DDBDRAFT_0206450 Kit ELISA, DDBDRAFT_0215015 Kit ELISA, DDB_0206450 Kit ELISA, DDB_0215015 Kit ELISA, TRA1 Kit ELISA, HSP90B1 Kit ELISA, ERp99 Kit ELISA, GRP94 Kit ELISA, TA-3 Kit ELISA, Targ2 Kit ELISA, Tra-1 Kit ELISA, Tra1 Kit ELISA, endoplasmin Kit ELISA, GP96 Kit ELISA, fb61d09 Kit ELISA, tra-1 Kit ELISA, wu:fb61d09 Kit ELISA, wu:fq25g01 Kit ELISA, Grp94 Kit ELISA, ECGP Kit ELISA, Gp96 Kit ELISA, ppk98 Kit ELISA, hsp108 Kit ELISA, heat shock protein 90kDa beta (Grp94), member 1 Kit ELISA, heat shock protein Hsp90 family protein Kit ELISA, 94 kD glucose-regulated protein Kit ELISA, heat shock protein 90 beta family member 1 Kit ELISA, heat shock protein 90, beta (Grp94), member 1 Kit ELISA, heat shock protein 90, beta (grp94), member 1 Kit ELISA, heat shock protein 90kDa beta family member 1 L homeolog Kit ELISA, hsp90b1 Kit ELISA, grp94 Kit ELISA, HSP90B1 Kit ELISA, Hsp90b1 Kit ELISA, hsp90b1.L Kit ELISA

Sujet: Heat shock proteins (HSPs), which are important for a number of different intracellular functions, are occasionally found on the surface of cells. The function of heat shock protein on the cell surface is not understood, although it has been shown to be greater in some tumor cells and some virally infected cells. Surface expression of both glycoprotein 96 (gp96) and Hsp70 occurs on tumor cells, and this expression correlates with natural killer cell killing of the cells. Glycoprotein 96 (gp96), also known as tumor rejection antigen 1 (TRA1), or glucose regulated protein 94 (GRP94), is a cell surface protein that binds adenosine. Thus, gp96 is also called adenotin and it is similar to various stress-induced proteins. The gp96 serves as a natural adjuvant for chaperoning antigenic peptides into the immune surveillance pathways. Glycoprotein 96 Can Chaperone Both MHC Class I- and Class II-Restricted Epitopes for In Vivo Presentation, but Selectively Primes CD8+ T Cell Effector Function gp96 facilitated the cross-presentation of a class I-restricted peptide and priming of effector function in cognate CD8 cells. Interestingly, gp96 also facilitated the in vivo presentation of a class II-restricted peptide, however, the resulting CD4 cell response did not involve the development of effector function. Taken together, these data suggest that gp96 is an inflammatory mediator that selectively primes CD8 cell effector function.

Pathways: Thyroid Hormone Synthesis, Activation of Innate immune Response, ER-Nucleus Signaling, Toll-Like Receptors Cascades