Practicals Flashcards
Which tracks are:
- sample cleaved with DNase
- undigested sample
- sample cleaved with Pvu II
- size markers
and why?
- undigested sample because it hasn’t moved very far due to the large molecular mass
- sample cleaved with Pvu II because this endonuclease cleaves the DNA in two places becuse it is specific
- sample cleaved with DNase becuase this endonuclease is non-specific so cleaves the DNA in a number of places. No bands are present because the molecular weights of each fragment are so small
- size markers.
How can you calculate the length of a DNA fragment in base pairs?
What does the gradient of the Lineweaver-Burk plot represent?
What is the role of ethidium bromide in PAGE?
It acts as a fluroescent tag
Why is there a second peak at 410nm in the elution profile of MetHb and GFP?
It is the ‘foothills’ of GFP absorbance that will peak at 490nm, since GFP has some absorbance at 410nm
How do you calculate turnover number?
What is the specificity of micrococcal nuclease?
Micrococcal nucelase demonstrates 30-fold preference for cleavage to the 5’ side of A or T rather than at G or C.
It preferentially cuts linker DNA (i.e. stretches without nucleosomes) to protect nucleosomal DNA.
What does the x-intercept represent on a Lineweaver-Burk plot?
What is the role of micrococcal nuclease digestion in the separation of histones?
It digests insoluble chromatin into insoluble oligonucleosomes
How would you calculate the amount of MetHb applied to the column?
Name two factors which affect the rate at which proteins move through a polyacrylamide gel?
- Molecular weight
- Presence of charged side chains
Why is DNA a polyanion?
Its phosphate groups have a high pK
What determines whether ion-exchange materials are anionic or cationic?
The nature of the ion they attract
How can you tell the difference between a digested and undigested sample on a polyacrylamide gel?
Undigested DNA is represented by a sharp band
Digested DNA is represented by a smear on the gel
How would you calculate the amount of MetHb recovered from the column?
What is the structure of genomic DNA in eukaryotes?
- Compacted into chromatin which contains a nucleosome
- Nucleosome consits of eight positively charged, small proteins (histones) and a piece of DNA wrapped around the histone octamer
- Histone octamer consits of two dimers of the proteins H2A and H2B and two dimers of H3 and H4
How is activity of beta-galactosidase measured?
By measuring ONP production from the hydrolysis of the artifical substrate ONPG
What is the first step when separating histone proteins?
Incubation and washing with a buffer to break the cell membranes and denature proteins not involved with histones
What is an endonuclease?
An enzyme that hydrolyses the phosphodiester backbone of DNA in interior positions
Which of the following methods can be used to elute proteins from an anionic exchange column?
a) Decrease the ionic strength of the mobile phase.
b) Increase the ionic strength of the mobile phase.
c) Increase the salt concentration of the mobile phase.
d) Decrease the pH of the mobile phase.
- *b) Increase the ionic strength of the mobile phase.
c) Increase the salt concentration of the mobile phase.
d) Decrease the pH of the mobile phase.**
How can you get protiens off affinity and ion-exchange columns?
By varying:
- salt concentration in the buffer
- the ionic strength of the buffer
- the pH of the buffer.
Why do we carry out micrococcal nuclease digestion at 37ºC and in the presence of CaCl2?
37ºC is the optimal temperature for the action of the micrococcal nuclease enzyme, and CaCl2 provides the Ca2+ ions the enzyme requires
What effect does a competitive inhibitor have on Vmax and KM?
Vmax – no change
KM – increased
What is the Beer-Lambert Law equation, rearranged to find concentration?