14. Molecular Technique and Diagnosis - Analysis of Proteins Flashcards
How are proteins separated in gel electrophoresis?
On the basis of size, shape or charge.
How does isoelectric focusing work in protein separation?
A stable pH gradient is established in a gel using an electric field. A protein solution is added and the electric field is reapplied. After staining, proteins distribute themselves along the pH gradient according to their pI values. When they reach pH=pI there is no net charge on the proteins so they stop migrating.
How does sodium dodecyl sulphate polyacrylamide gel electrophoresis work?
It separates proteins according to their size and molecular weight. The protein is denatured and reverted back to primary structure and put through gel electrophoresis.
How does two-dimensional electrophoresis (2-D PAGE) work?
It separates complex mixtures of proteins. Then compares results to normal state to diagnose disease states.
How are proteins identified in proteomics?
Digest the protein with trypsin and perform mass spectrometry. generate a list of peptide sizes and compare to the database of predicted peptide sizes for known proteins to identify the protein.
What is proteomics?
Analysis of all proteins expressed from a genome.
What is molecular diagnosis?
Analysis of a single purified protein.
What are polyclonal antibodies?
Produced by many B lymphocytes and have multiple different antibodies. They’re specific to one antigen but have multiple epitopes (bit recognised by the immune system).
How are polyclonal antibodies collected?
Inject the antigen into an animal over three to four weeks on several occasions to get an immune reasons. Isolate the polyclonal antibodies.
What are monoclonal antibodies?
Produced by one B lymphocyte and have one identical antibody. They’re specific to one antigen and have one epitope.
How do you make monoclonal antibodies?
Take spleen cells from infected animals and myeloma cells from a cell culture and fuse them in polyethylene glycol. Select and grow hybrid cells and then select cells the make the desired monoclonal antibody. Then either grow them in a mass culture, freeze thaw them or inject them into animals to induce a tumour and take the antibodies that way.
How is western blotting used to detect proteins?
Gel electrophoresis results are transferred to a more solid source of nitrocellulose replica. An antibody specific to the one protein of interested is added. A second antibody specific to the first antibody is added and if it binds it shows the antibody of interest was present. This is then transferred to an immunoblot.
He does enzyme-linked immunoabsorbant assay (ELISA) work?
There is an antigen-coated well which is washed so a specific antibody binds to the antigen. It’s washed again and enzyme-linked antibodies bind to the specific antibody. A final wash is done and the substrate is added and converted by the enzyme into the coloured product. The rate of colour formation is proportional to the amount of specific antibody.
How are the following plasma hormones detected and assay studied?
a. Cortisol
b. Insulin
c. TSH
d. T3/T4
a. RIA
b. RIA or ELISA
c. RIA or ELISA
d. RIA or ELISA
What are two continuous assay used to measure the product?
Spectrophotometery and chemoluminescence.
