long question (12) Flashcards
1
Q
describe and compare techniques used to detect protein expression
A
2
Q
describe techniques used for protein expression
A
- western blotting
- ELISA
3
Q
what is western blotting
A
- used for separating & identifying proteins
- checks protein expression in a mixture of proteins
4
Q
western blotting steps
A
- mixture of proteins are separated based on native charge through gel electrophoresis & molecular weight that are denatures or native
- electroblotting of the separated proteins onto a solid membrane
- transferred to a membrane producing a band for each protein
- detection of target proteins by specific antibodies e.g. flu Ab
5
Q
western blotting advantages
A
- the ability to indentify specific target proteins & molecular weight
- can be used for many purposes as an effective early diagnostic tool
- used to detect changes in protein expression
6
Q
western blotting disadvantages
A
- time-consuming
- high demand in terms of experience
- difficulties in reproducibility
- requires optimising the experimental conditions (i.e. protein isolation, buffers, gel concentration)
7
Q
what is ELISA
A
- enzyme-linked immunosorbent assay
- designed for detecting and quantifying soluble substances in a complex mixture such as proteins, antibodies etc.
8
Q
ELISA steps
A
- labelled antibodies are used to quantify target molecules
- chemiluminiscent emit light
- chemifluorescent is the emission of photons
- colorimetric is the formation of solubule colorful substances
9
Q
ELISA advantages
A
- simple procedure
- high specificity and sensitivity
- high efficiency
- generally safe and eco-friendly
- cost-effective
10
Q
ELISA disadvantages
A
- high possibility of false positive & negatives
- antibody instability
- labor intensive and antibody prep is expensive
- expensive culture media required