Measuring Proteins and Genes

Question 1

What is spectrophotometry?

Answer 1

• many compounds in a solution absorb visible light in proportion to their concentration
• while many biomolecules are colourless, combining with a reagent produces a coloured compound

Question 2

How do you quantify nucleic acids?

Answer 2

• nucleic acids (DNA and RNA) absorbs ultraviolet light at a wavelength of 260nm
• commonly used to check the purity of isolated nucleic acid

Question 3

How do you measure total protein abundance via spectrophotometry?

Answer 3

• protein is mixed with a dye called Coomassie blue
• in acidic conditions this results in a colour change brown-blue
• protein quantity can be detected at 595nm

Question 4

How do you completed qRT-PCR?

Answer 4

• sample collection
• RNA extraction
• reverse transcription
• RT-PCR amplification
• results

Question 5

What is the western blot?

Answer 5

• the most widely used lab technique to detect ‘specific’ protein in a tissue homogenate or protein extract
• protocols:
  
  • sample collection
  • protein extraction
  • electrophoresis to seperate protein size
  • electrotransferring protein from gel to membrane
  • antibody probing and detection

Question 6

What is Enzyme Linked Immunosorbent Assay (ELISA)?

Answer 6

• capture antibody binds to well
• add sample
• wash microplate
• add detection antibody
• wash microplate
• add substrate
• read microplate
• calculate results