Lecture 6 Flashcards
(48 cards)
Are large or small peaks preferred in a protein vs fraction graph? Why?
Small.
Shows highly enriched as opposed to lack of purification.
If you recover 100 units of activity in 100µg of protein, what is the specific activity?
If you recover 80 units of activity in 2µg of protein, what is the specific activity?
Which is preferred, and why?
1 unit/µg.
40 units/µg.
Latter preferred, because less total activity, but much purer.
What 4 things must a useful protein assay be?
Specific - only measuring activity of interest.
Sensitive - use only small quantity of protein.
Accurate - requiring correct conditions for activity.
Convenient.
Three types of assay for protein detection?
Immunological detection,
Enzyme activity,
Functional essay (bioassay).
If a protein has enzyme activity, what type of assay should you use?
Enzyme activity assay.
If a protein has no enzyme activity, but has an antibody available, which assay should you use?
Immunoassay.
If a protein has no enzyme activity, and has no antibody available, which assay should you use?
Bioassay.
3 positives of using immunological detection?
Very sensitive,
Specific,
Can be applied to almost any protein (and other biological molecules).
1 negative of using immunological detection?
Usually requires prior purification of protein to generate antibody.
What is an ELISA?
Enzyme-Linked ImmunoSorbent Assay.
What apparatus is an ELISA performed in? Why is this beneficial in terms of throughput?
Microtitre tray.
Can perform many simultaneously.
How does an ELISA work?
Detection antibody binds with target protein. Labelled antibody then binds to detection antibody, producing a coloured product.
What is a common reporter used in yeast and bacterial genetics?
ß-galactosidase.
What does the X-gal substrate enable in a direct enzyme activity assay?
Colour selection depending on the uptake of an enzyme.
What is a disadvantage to using traditional ß-galactosidase (lacZ) assay?
Blue product insoluble so can’t use a spectrometer and therefore difficult to quantify.
How can a colorimetric assay be performed on ß-galactosidase? And how does it work?
ONPG used as substrate, catalysed by ß-galactosidase to form galactose and o-Nitrophenol (yellow). Absorbance measured at 420nm. Rate of increase of A420 implies enzyme activity.
How can a fluorimetric assay be performed on ß-galactosidase?
4-methylumbelliferyl ß-D-galactopyranoside (MUG) converted via ß-galactosidase to form 4-methylumbelliferone (4-MU). UV light (365nm) shone on 4-MU, with blue light (455nm) being returned.
Rate of increase of emission of light = Enzyme activity.
When glucose-6-phosphate reacts with the enzyme Glucose-6-P dehydrogenase in a direct enzyme activity assay, what is formed, and what is the enzyme’s cofactor?
6-Phosphogluconolactone.
NAD+.
What is detected in direct enzyme activity assay of glucose-6-phosphate? How does it help calculate enzyme activity?
340nm UV light absorbance detected.
Rate of increase of A340 = enzyme activity.
Give an example of a coupled enzyme activity assay with glucose as the starting product.
Glucose reacts with Hexokinase enzyme w/ ATP cofactor, producing Glucose-6-phosphate.
Glucose-6-phosphate reacts with Glucose-6-P-dehydrogenase with NAD+ as a cofactor, forming 6-phosphogluconolactone.
What is a continuous assay?
Change in absorbance/fluorescence can be measured continuously over time.
What is a stopped assay?
An assay with a single end point measurement.
Why are stopped assays occasionally preferred?
Usually because measurement of product is more difficult, e.g. requires HPLC, measurement of radio labelled reactants, etc.
What type of assay is not performed in a test-tube/biochemically?
Bioassays.
