Cell Prac

Question 1

What do you need to know about a protein for a western blot

Answer 1

• its antibody, or a C/N-terminal tag antibody and it’s secondary antibody (which type of SDS-page wrt% acrilimyde do you run pre-nitrogencellulose immunoblot)
• what size it is - where do you look for it?

Question 2

SDS page

Answer 2

separates protein into suitable masses

Question 3

How do you run the experiment:

Answer 3

1) cell lysis and total protein extraction
2) SDS page
3) immublot
4) Ab probing
5) positive control = purified protein
6) negative control = don’t transform the cells

Question 4

Mutations preventing catalysis

Answer 4

• bp addition/deletion/substitution that introduces an early stop codon
• amino acid that creates steric encumbrance, prevents correct folding of other parts of the protein
• mutation in the promoter or ribosome binding sites that prevent transgene transcription and translation, respectively
• amino acid substitutions that impact substrate affinity

Question 5

When asked to analyse an electrophoresis output

Answer 5

• where is the protein you are looking for and why
• what are the other things? they must be recognised by the same antibody (esp. if polyclonal). how do they behave?

Question 6

why might clearly less proteins show up in the Ponceau gel?

Answer 6

• mistake in protein quantification
• loss during gel loading
• what you’re looking for isn’t there

Question 7

Additional controls?

Answer 7

• truncated mutants would have a lower weight band
• 5’UTR, ribosome binding site reduces protein amounts, or its activity; same weight but lower intensity

Question 8

Lower weight on the kDA

Answer 8

proteins made the enzyme?

Question 9

Secondary antibodies or

Answer 9

• conjugate detection system (such as fluorescent tags)
• dual flourescence at N and C termini show their distinct fates

Question 10

What would you see in electrophoresis?

Answer 10

an intact protein and a fragment at lower weight

Question 11

fluorescence microscopy

Answer 11

• sub cellular localisation and fate analysis
• what happens to it? is it cleaved? stabilised?

Question 12

If it is stabilised in active form?

Answer 12

a dominant mutation that results in truncation; sub-cellular localisation to nucleus is constitutive

Question 13

if it doesn’t bind to DNA

Answer 13

• is it truncated before it’s binding site
• is it no longer recognised by its proteins