EGFR Kit ELISA

N° du produit ABIN1379962

Détail du produit EGFR Kit ELISA

Antigène: EGFR (Epidermal Growth Factor Receptor (EGFR))

Reactivité: Humain

Méthode de détection: Colorimetric

Type de méthode: Sandwich ELISA

Gamme de detection: 32-2000 pg/mL

Seuil minimal de détection: 32 pg/mL

Application: ELISA

Fonction: The OmniKine? Human EGFR ELISA Kit contains the components necessary for quantitative determination of natural or recombinant Human EGFR concentrations within any experimental sample including cell lysates, serum and plasma. This particular immunoassay utilizes the quantitative technique of a "Sandwich" Enzyme-Linked Immunosorbent Assay (ELISA) where the target protein (antigen) is bound in a "sandwich" format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator. The capture antibodies coated to the bottom of each well are specific for a particular epitope on Human EGFR while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of non- specific binding between proteins to other proteins or to the solid phase. After incubation and "sandwiching" of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody (either covalently or via Avidin/Streptavidin-Biotin interactions), allowing for a colorimetric reaction to ensue upon substrate addition. When the substrate TMB (3, 3', 5, 5'-Tetramethylbenzidine) is added, the reaction catalyzed by peroxidase yields a blue color that is representative of the antigen concentration. Upon sufficient color development, the reaction can be terminated through addition of Stop Solution (2 N Sulfuric Acid) where the color of the solution will turn yellow. The absorbance of each well can then be read by a spectrophotometer, allowing for generation of a standard curve and subsequent determination of protein concentration.

Marque: OmniKine™

Type d'échantillon: Cell Lysate, Serum, Plasma

Analytical Method: Quantitative

Specificité: The Human EGFR ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human EGFR proteins.

Réactivité croisée (Details): The Human EGF-R ELISA is capable of recognizing both recombinant and naturally produced Human EGF-R proteins. The antigens listed below were tested at 50 ng/mL and exhibited less than 3% cross reactivity. Murine: EGF-R The antigens listed below were tested at 50 ng/mL and did not exhibit significant cross reactivity or interference. Human: EGF, ErbB2, ErbB3

Attributs du produit: The Human EGFR ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human EGFR proteins within the range of 32-2000 pg/mL.

Ingrédients:

• Microstrips Coated w / Capture Antibody: 12 x 8-Well Microstrips
• Protein Standard: Lyophilized (100 ng), Red container
• Biotinylated Detection Antibody: Lyophilized, Yellow container
• 400x Streptavidin-HRP: 30 μL, Blue container
• Wash Buffer (10x): 50 mL, Clear containter
• Assay Diluent: 50 mL, Clear container
• Ready-to-Use Substrate: 12 mL, Brown container
• Stop Solution: 12 mL, Clear container
• Adhesive Plate Sealers: 4 Sheets
• Technical Manual 1 Manual

Matériel non inclus: The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
Microplate reader able to measure absorbance at 450 nm (with correction wavelength set to 540 nm or 570 nm)
Micropipettes with capability of measuring volumes ranging from 1 μl to 1 mL
Deionized or sterile water
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent paper or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 mL
Bench
top centrifuge (optional)
Bench
top vortex (optional)
Orbital shaker (optional)

Information d'application

Plaque: Pre-coated

Protocole: This particular immunoassay utilizes the quantitative technique of a Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) where the target protein (antigen) is bound in a sandwich format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator. The capture antibodies coated to the bottom of each well are specific for a particular epitope on the Human EGF-R cytokine while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of non-specific binding between proteins to other proteins or to the solid phase. After incubation and sandwiching of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody (either covalently or via Avidin/Streptavidin-Biotin interactions), allowing for a colorimetric reaction to ensue upon substrate addition. When the substrate TMB (3, 3’, 5, 5’- Tetramethylbenzidine) is added, the reaction catalyzed by peroxidase yields a blue color that is representative of the antigen concentration. Upon sufficient color development, the reaction can be terminated through addition of Stop Solution (2 N Sulfuric Acid) where the color of the solution will turn yellow. The absorbance of each well can then be read by a spectrophotometer, allowing for generation of a standard curve and subsequent determination of protein concentration.

Préparation de l'échantillon:

If samples are to be used within 24 hours, aliquot and store at 4 °C. If samples are to be used over a long period of time, aliquot and store between -20 °C and -80 °C, depending on the duration of storage.
Note: Samples containing a visible precipitate or pellet must be clarified prior to use in the assay.
Caution: Avoid repeated freeze/thaw cycles to prevent loss of biological activity of proteins in experimental samples.

• Cell Lysate and Supernatants:
  Remove large cell components via centrifugation and perform the assay. Cell lysates and supernatants require a dilution using Assay Diluent. A serial dilution may be performed to determine a suitable dilution factor for the sample. For future use of the sample, follow the sample storage guidelines stated above.
• Serum:
  Allow samples to clot in a serum separator tube (SST) for 30 minutes. After sufficient clotting, centrifuge at 1000 x g for 15 minutes and remove serum from SST in preparation for the assay. Serum samples require at least a 1:50 dilution using Assay Diluent. For future use of the sample, follow the storage guidelines above.
• Plasma:
  Use heparin, citrate or EDTA as an anticoagulant to gather plasma from original biological sample. After collection of the plasma, centrifuge for 15 minutes at 1000 x g. This step must be performed within 30 minutes of plasma collection. Plasma samples require at least a 1:50 dilution using Assay Diluent. Afterwards, perform the assay or for future use of the sample, follow the storage guidelines stated above.

Procédure de l'essai:

Note: If possible, all incubation steps should be performed on an orbital shaker to equilibrate solutions when added to the microplate wells. Also, all provided solutions should be at ambient temperature prior to use.
Note: Avoid adding solutions into wells at an angle, always keep pipette tip perpendicular to plate bottom.

Reconstitution of Provided Materials:

1. Reconstitute the Biotin-Conjugated Detection Antibody in 67 µL of ddH₂O for a concentration of 180 µg/ml.
2. Reconstitute the Protein Standard in 100 µL of ddH₂O for a concentration of 340 ng/ml.
3. Dilute the 50 mL of 10x Wash Buffer in 450 mL of ddH2O for 500 mL of 1x Wash Buffer.

Addition of Known Standard and Unknown Sample to Immunoassay:

The OmniKine™ Human CD163 ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human CD163 proteins

Calcul des résultats:

Generation of Standard Curve and Interpretation of Data
1. Average the duplicate or triplicate readings for each standard, control and sample and subtract the average zero standard optical density.
2. Generate a standard curve by using Microsoft Excel or other computer software capable of establishing a 4- Parameter Logistic (4-PL) curve fit. If using Excel or an alternative graphing tool, plot the average optical density values in absorbance units (y-axis) against the known standard concentrations in pg/ml (x-axis). Note: Only use the values in which a noticeable gradient can be established. Afterwards, generate a best fit curve or trend-line through the plotted points via regression analysis.

Restrictions: For Research Use only

Stockage

Précaution d'utilisation: Reagents provided in this kit may be harmful if ingested, inhaled or absorbed through the skin. Please carefully review the MSDS for each reagent before conducting the experiment.
Stop Solution contains 2 N Sulfuric Acid (H2SO4) and is an extremely corrosive agent. Please wear proper eye, hand and face protection when handling this material. When the experiment is finished, be sure to rinse the plate with copious amounts of running water to dilute the Stop Solution prior to disposing the plate.

Conseil sur la manipulation: This ELISA kit is intended for research purposes only, NOT diagnostic or clinical procedures of any kind.
Materials included in this kit should NOT be used past the expiration date on the kit label.
Reagents or substrates included in this kit should NOT be mixed or substituted with reagents or substrates from any other kits.
Variations in pipetting technique, washing technique, operator laboratory technique, kit age, incubation time or temperature may cause differences in binding affinity of the materials provided.
The assay is designed to eliminate interference and background by other cellular macromolecules or factors present within any biological samples. However, the possibility of background noise cannot be fully excluded until all factors have been tested using the assay kit.

Reagents provided in this kit may be harmful if ingested, inhaled or absorbed through the skin. Please carefully review the MSDS for each reagent before conducting the experiment.
Stop Solution contains 2 N Sulfuric Acid (H2SO4) and is an extremely corrosive agent. Please wear proper eye, hand and face protection when handling this material. When the experiment is finished, be sure to rinse the plate with copious amounts of running water to dilute the Stop Solution prior to disposing the plate.

Stock: 4 °C

Stockage commentaire: Note: If used frequently, reagents may be stored at 4 °C.

• Unopened Kits: Store at 4 °C for 6 months.
• Microstrips Coated w/ Capture Antibody, 400x Streptavidin-HRP Wash Buffer (10x), Assay Diluent Ready-to-Use Substrate, Stop Solution: 6 Months at 4 °C
• Protein Standard, Biotinylated Detection Antibody: Lyophilized: 6 Months (if Reconstituted: 1 Month) at 4 °C

Détail du antigène

Antigène: EGFR (Epidermal Growth Factor Receptor (EGFR))

Autre désignation: EGFR (EGFR Produits)

Synonymes: C-erb Kit ELISA, CG10079 Kit ELISA, D-EGFR Kit ELISA, D-Egf Kit ELISA, DEGFR Kit ELISA, DER Kit ELISA, DER flb Kit ELISA, DER/EGFR Kit ELISA, DER/faint little ball Kit ELISA, DER/top Kit ELISA, DER/torpedo Kit ELISA, DER1 Kit ELISA, DEgfr Kit ELISA, Degfr Kit ELISA, Der Kit ELISA, DmHD-33 Kit ELISA, Dmel\\CG10079 Kit ELISA, EFG-R Kit ELISA, EGF-R Kit ELISA, EGFR Kit ELISA, EGFr Kit ELISA, EGfr Kit ELISA, EK2-6 Kit ELISA, Egf Kit ELISA, Egf-r Kit ELISA, EgfR Kit ELISA, El Kit ELISA, Elp Kit ELISA, Elp-1 Kit ELISA, Elp-B1 Kit ELISA, Elp-B1RB1 Kit ELISA, HD-33 Kit ELISA, TOP Kit ELISA, Torpedo/DER Kit ELISA, Torpedo/Egfr Kit ELISA, c-erbB Kit ELISA, d-egf-r Kit ELISA, dEGFR Kit ELISA, dEGFR1 Kit ELISA, dEgfr Kit ELISA, der Kit ELISA, egfr Kit ELISA, flb Kit ELISA, l(2)05351 Kit ELISA, l(2)09261 Kit ELISA, l(2)57DEFa Kit ELISA, l(2)57EFa Kit ELISA, l(2)57Ea Kit ELISA, mor1 Kit ELISA, top Kit ELISA, top/DER Kit ELISA, top/flb Kit ELISA, torpedo/Egfr Kit ELISA, torpedo/egfr Kit ELISA, EGFR12 Kit ELISA, EGFR15 Kit ELISA, egfr1 Kit ELISA, Erbb2 Kit ELISA, ERBB Kit ELISA, ERBB1 Kit ELISA, HER1 Kit ELISA, PIG61 Kit ELISA, mENA Kit ELISA, ErbB-1 Kit ELISA, Errp Kit ELISA, 9030024J15Rik Kit ELISA, AI552599 Kit ELISA, Erbb Kit ELISA, Errb1 Kit ELISA, Wa5 Kit ELISA, wa-2 Kit ELISA, wa2 Kit ELISA, epidermal growth factor receptor Kit ELISA, Epidermal growth factor receptor Kit ELISA, epidermal growth factor receptor a (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian) Kit ELISA, EGFR Kit ELISA, Egfr Kit ELISA, egfra Kit ELISA, egfr1 Kit ELISA, LOC5564544 Kit ELISA

Sujet: Human EGF receptor is not only a cell surface receptor for EGF, but also for other members of the EGF family, such as TGF-alpha, BTC/betacellulin, AREGAREGB/amphiregulin, HBEGF, GP30 and vaccinia virus growth factor. Ligand binding triggers a conformation change, leading to activation of the kinase and subsequent phosphorylation of down-stream protein kinases. Hence, EGF receptor is involved in the control of cell growth, proliferation and differentiation. Such receptor also phosphorylates MUC1 in breast cancer cells and increases the interaction of MUC1 with SRC and CTNNB1/beta-catenin. EGF binding triggers dimerization of the receptor and promotes auto- phosphorylation. The activate receptor dimer binds one EGF molecule while the heterodimer binds with ERBB2 and interacts with ERRFI1. Interaction with ERRFI1 inhibits dimerization of the kinase domain and auto-phosphorylation. ERRFI1 bounded EGF receptor also binds with RIPK1 and interacts with CBL via the auto-phosphorylated C-terminal tail. EGF receptor is part of a complex with ERBB2 and either PIK3C2A or PIK3C2B that may indirectly interact in the auto-phosphorylated form with PIK3C2B. Upon EGF treatment, the ligand bounded receptor complex also interacts with PELP1, binds with MUC1, interacts with AP2M1, interacts with GAB2, and interacts with COPG. These interactions are essential for regulation of EGF-dependent nuclear transport of EGFR by retrograde trafficking from the Golgi to the ER. In addition, EGF receptor interacts with FER and TNK2. Such interaction is dependent on EGF stimulation and kinase activity of EGFR. EGF receptor phosphorylation of Ser-695 is partial and occurs only if Thr- 693 is phosphorylated. Monoubiquitinated and polyubiquitinated occur upon EGF stimulation, which does not affect tyrosine kinase activity or signaling capacity, but may play a role in lysosomal targeting. Polyubiquitin linkage is mainly through 'Lys-63' while linkage through 'Lys-48', 'Lys-11' and 'Lys-29' also occur. Studies have shown that defects in EGFR are associated with lung cancer. Source: Entrez Gene, Swiss-Prot

Pathways: Signalisation NF-kappaB, Signalisation RTK, Fc-epsilon Receptor Signaling Pathway, EGFR Signaling Pathway, Neurotrophin Signaling Pathway, Stem Cell Maintenance, Hepatitis C, Positive Regulation of Response to DNA Damage Stimulus, Interaction of EGFR with phospholipase C-gamma, Thromboxane A2 Receptor Signaling, EGFR Downregulation, S100 Proteins