Standard and Sample Diluent Buffer: 1X PBS containing 0.5-3% BSA
Antibody and HRP-Conjugate Diluent Buffer: 1X PBS containing 0.5-3% BSA
Stop Solution: 5% Sulfuric Acid
Target
Epidermal Growth Factor Receptor (EGFR)
Research Area
Cytokines, Tumor Immunity, Infection Immunity, Cardiovascular Biology, Cancer
Reactivity
Pig
Tested Applications
ELISA
Recommended dilutions
Capture Antibody: 1/500 - 1/1000, Biotin-conjugated Detection Antibody: 1/500 - 1/1000, HRP-Conjugate: 1/500 - 1/1000. Optimal dilutions/concentrations should be determined by the end user.
Reconstitution
Reconstitute the standard with 1 ml of Standard Diluent to obtain a stock standard solution of 5000 pg/ml. Label tubes in preparation for the serial dilutions (2500, 1250, 625, 312.5, 156.25, and 78.13 ng/ml). Aliquot 0.5 ml of the Standard Diluent into each tube. Add 0.5 ml of the highest standard solution into the 1st tube and mix thoroughly. Transfer 0.5 ml from the 1st to 2nd tube, mix thoroughly, and so on.
Storage
Aliquot and store at -20°C in the dark. Avoid repeated freeze/thaw cycles.
Analyse immediately or store samples at 2-8°C (within 24 hrs). For long term storage, aliquot and store at -20°C or -80°C. Avoid multiple freeze-thaw cycles.
Serum: Samples should be collected into a serum separator tube. Coagulate the serum by leaving the tube undisturbed in a vertical position overnight at 4°C or at room temperature for up to 1 hr. Centrifuge at approximately 1000 × g for 20 mins. If precipitate appears, centrifuge again. Assay immediately or aliquot and store at -20°C or -80°C.
Plasma: Collect plasma using EDTA as an anticoagulant. Centrifuge for 15 mins at 1000 × g, within 30 mins of collection. If precipitate appears, centrifuge again. Avoid hemolytic samples.
Availability
Shipped within 5-10 working days.
Note
THIS PRODUCT IS FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC, THERAPEUTIC OR COSMETIC PROCEDURES. NOT FOR HUMAN OR ANIMAL CONSUMPTION.
Directions for use
Bring all components to room temperature (18-25°C) and briefly spin or centrifuge the vials before use. Working solutions should be prepared and used immediately.
Recommended Procedure:
Dilute the Capture Antibody to working concentration using Coating Buffer. Immediately coat the 96-well plate with diluted Capture Antibody (100 µl per well). Seal the plate and incubate at 2-8 °C overnight.
Remove the liquid from each well. Do not wash. Block the plate with Blocking Buffer (200 µl per well) at 37 °C for 1 hour.
Remove the liquid from each well. Do not wash. Either proceed with the following steps immediately or dry the plate at 37 °C for 30 minutes, then store at -20 °C with dessicant for up to 6 months.
Add 100 µl of standards or sample into the appropriate wells. Cover with a plate sealer and incubate at 37 °C for 1.5 hours.
Remove the liquid from each well. Do not wash. Add appropriately diluted Biotin-Conjugated Detection Antibody (100 µl per well). Cover the plate with a new plate sealer and incubate at 37 °C for 1 hour.
Remove the liquid from each well. Wash with Wash Buffer (350 µl per well) and allow to soak for 1-2 min. Remove the liquid by inverting and tapping the plate on to absorbent paper. Repeat the wash process 3 times.
Add appropriately diluted HRP-Conjugate (100 µl per well). Cover the plate with a new plate sealer and incubate at 37 °C for 30 min.
Repeat the wash process in Step 6, for a total of 5 times.
Add Substrate Solution (90 µl per well). Cover the plate with a new plate sealer and incubate at 37 °C for 15-30 min. Keep the plate in the dark and avoid exposure to light.
Add Stop Solution (50 µl per well). Tap the side of the plate to ensure thorough mixing.
Measure the absorbance immediately using a microplate reader set at 450 nm.
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