Immunoglobulin Y (abbreviated as IgY) is a type of immunoglobulin which is the major antibody in bird, reptile, and lungfish blood. It is also found in high concentrations in chicken egg yolk. As with the other immunoglobulins, IgY is a class of proteins which are formed by the immune system in reaction to certain foreign substances, and specifically recognize them. Horseradish peroxidase (HRP) conjugates are prepared by a modified Nakane and Kawaoi procedure (J. Histochem. Cytochem. 1974. 22, 1084). Peroxidase conjugates are commonly used for immunohistochemistry, Western blotting, and ELISA. Affinity-purified anti-horseradish peroxidase and conjugates are available for detection of horseradish peroxidase antigen or for signal amplification of HRP-containing reagents. For immunostaining of mammalian cells, an advantage of using anti-horseradish peroxidase is reduced background, since the antibody does not recognize the endogenous peroxidase-like enzymes found in those cells.
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Western blot analysis of SOX2 on different lysates with Chicken anti-SOX2 antibody (HA601499) at 1/5,000 dilution.
Lane 1: NCCIT cell lysate Lane 2: F9 cell lysate
Lysates/proteins at 15 µg/Lane.
Predicted band size: 34 kDa Observed band size: 36 kDa
Exposure time: 40 seconds; ECL: K1801;
4-20% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA601499) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat anti-Chicken IgY - HRP Secondary Antibody (HA1146) at 1/50,000 dilution was used for 1 hour at room temperature.
Direct ELISA analysis of Goat anti-Chicken IgY was performed by coating wells of a 96-well plate with 50 µl per well of Chicken IgY / Rabbit IgG / Mouse IgG / Human IgG / Guinea pig IgG diluted in carbonate/bicarbonate buffer, at a concentration of 1 µg/mL overnight at 4℃. Wells of the plate were washed, blocked with 1%BSA blocking buffer, and incubated with 100 µl per well of Goat anti-Chicken IgY polyclonal antibody (HA1146) diluted at a concentration of 250 ng/mL for 1 hours at room temperature. Detection was performed using an Ultra TMB Substrate for 10 minutes at room temperature in the dark. The reaction was stopped with sulfuric acid and absorbances were read on a spectrophotometer at 450 nm.
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