ELISAtoMeasureCytochromeP450ProteinConcentration.pptVIP

ELISA to Measure Cytochrome P450 Protein Concentration by: William Collins Objectives To develop an ELISA procedure to measure Cytochrome P450 protein. What Is An ELISA? E- Enzyme L- Linked I- Immuno S- Sorbent A- Assay This technique is designed to provide an ultra-sensitive process with dependable results. It uses a 96-well plate to measure a protein or substance based on an antigen/antibody reaction. Steps Involved in an ELISA Bind the protein or antigen to the plate. Then you block the plate to get rid of any non-specific binding sites. Incubate with the primary antibody which is specific for the antigen. Secondary antibody that is linked with an Enzyme is allowed to bind with the primary antibody. Use a Substrate for the enzyme which will cause color to be released. Sulphan Blue Results Cytochrome P450 Cytochrome P450 is a large group of enzymes that are found in the liver of mammals. They are the main step in the elimination and transformation of foreign substances. Abbreviations uL- microliters FBS- Fetal Bovine Serum PBS- Phosphate Buffered Saline TBS- Tris Buffered Saline nm- nanometers Microsomes We removed the liver from a normal rat and from a Phenobarbital treated rat. Use a Potter-Eljeham homogenizer at 1000 RPM to create a homogenate. Centrifuge the homogenate at 600g for 10 minutes to produce a crude homogenate. Centrifuge the remaining supernatant at 15,000g for 1 hour to separate out the mitochondrial pellet. Centrifuge the remaining supernatant at 100,000g for 1 hour to yield the microsomal pellet. ELISA Procedure Add 100 uL protein to plate wells in triplicate. Add 100 uL of 2x Carbonate-Bicarbonate buffer to each well. Cover and store overnight at 4°C. Add 200 uL of 50% FBS in PBS to each well. Mix for 1 hour. This is the blocking solution. Wash plate out with TBS-Tween 3 times Add 200uL Primary Antibody Solution to each well. Mix for 1hour at 37oC Wash plate out with TBS-Tween 3 times. Add 200ul Secondary Antibody Solution to each wel