long question (12)

Question 1

describe and compare techniques used to detect protein expression

Question 2

describe techniques used for protein expression

Answer 2

• western blotting
• ELISA

Question 3

what is western blotting

Answer 3

• used for separating & identifying proteins
• checks protein expression in a mixture of proteins

Question 4

western blotting steps

Answer 4

• 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

Question 5

western blotting advantages

Answer 5

• 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

Question 6

western blotting disadvantages

Answer 6

• time-consuming
• high demand in terms of experience
• difficulties in reproducibility
• requires optimising the experimental conditions (i.e. protein isolation, buffers, gel concentration)

Question 7

what is ELISA

Answer 7

• enzyme-linked immunosorbent assay
• designed for detecting and quantifying soluble substances in a complex mixture such as proteins, antibodies etc.

Question 8

ELISA steps

Answer 8

• 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

Question 9

ELISA advantages

Answer 9

• simple procedure
• high specificity and sensitivity
• high efficiency
• generally safe and eco-friendly
• cost-effective

Question 10

ELISA disadvantages

Answer 10

• high possibility of false positive & negatives
• antibody instability
• labor intensive and antibody prep is expensive
• expensive culture media required