Measuring proteins and genes

Question 1

spectophotometry-
- what is the principle
- how to make colourless molecules colourful
- what does nanodrop instrument measure
- example assays to standardise amounts of protein measured to allow comparison

Answer 1

• molecules absorb light in proportion to their concentration. more light absorbed, greater conc
• combine with regent which allows assessment
• quantitation of nucleic acids by knowing they absorb UV at a wavelength of 260 nm
• the Bradford, BCA, and lowry assay

Question 2

polymerase chain reaction test-
- process
- how can you quantify how much DNA is in your sample

Answer 2

• take a sample
• extract RNA by enzymes
• add reverse transcriptase and free nucleotides to create complementary DNA.
• Add heat to break hydrogen bonds
• add heat resistant DNA polymerase to amplify DNA
• continue repetition of this
• use dyes which specifically signal/fluoresce when bound to specific DNA

Question 3

how to develop specific antibodies which can be used to measure specific proteins
- what people do in practice
- process of getting the antibodies using an animal

Answer 3

• buy antibodies to save time and its easy
• Inject antigen into host species, often a rabbit commercially
• Rabbit will respond to that antigen and generate antibodies against it
• Can separate the blood and serum
• add the same antigen to some beads
• mix the serum containing the antibodies with the beads and antibodies specific to the antigen bind to the beads
• Wash everything away and we are left with antigen-specific antibodies

Question 4

how to use the antibodies to measure specific proteins
- western blot protocol

Answer 4

○ Collect sample
○ Extract protein via homogenization and measure how much there is, eg. Use Bradford assay
○ Electrophoreses to separate protein size (large proteins at top, small at the bottom)
○ Electrotransfering protein from gel to blotting membrane
- Use antibody probing and detection - add primary antibody that’s very specific to target protein, add secondary antibody comp to primary antibody with detection signal on it, often light so we can measure light and therefore how much protein is on the membrane

Question 5

how to use the antibodies to measure specific proteins
- ELISA test (enzyme linked immunosorbent assay)

Answer 5

○ Capture antibody binds/stuck to well
○ Add sample (should bind if protein of interest present)
○ Wash microplate
○ Add detection antibody specific to protein of insulin
○ Wash microplate
○ Add substrate (shows detection signal)
- Read microplate and calculate results