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Please can you provide me with a protocol how to use the oncostatin ab 9633 in a direct elisa system. Which second ab do you recoment and how do you detect the protein. i understand that you use ab9633 as the capture ab, but what do you use as the detection ab?
Asked on Dec 01 2003
Thank you for your enquiry. Below is a general Direct ELISA protocol which should be helpful for you. If you have any more questions, please contact us again. Direct ELISA: To detect hOncostatin-M by direct ELISA (using 100µl/well antibody solution) a concentration of at least 0.5µg/ml of this antibody is required. This antigen affinity purified antibody, in conjunction with compatible secondary reagents (located at the bottom of the datasheet), allows the detection of 0.2 - 0.4 ng/well of recombinant hOncostatin-M. 1. Dilute Ag into coating buffer (1 x PBS, pH 7.4). Add 100 ul/well. Incubate overnight at 4C. 2. Wash plate with wash buffer (1 x PBS, 0.05% Tween-20). Add 300 ul/well of blocking buffer ( 1 x PBS, 0.05% Tween-20, 1% BSA). Incubate for 2hrs at room emp. 3. Wash plate with wash buffer. Dilute detecting Ab (biotinylated antibody) to 1 ug/ml in diluting buffer (1 x PBS, 0.05% Tween-20, 1% BSA). Add 100 ul/well of detecting antibody. Incubate for 2 hrs at room temp. 4. Wash plate with wash buffer. Dilute Avidin Peroxidase 1:2000 in dilutent. Add 100 ul/well. 5. Wash plate with wash buffer. Add 100 ul/well of ABTS substrate solution (10.5 Citric Acid, 150 mg ABTS in 500 ml, pH to 4.35). Add 100 ul/well of ABTS substrate solution. 6. Read absorbance at 405 nm.
Answered on Dec 03 2003