Flashcards in Biotech lab 3-background Deck (22)
What does the electrophoresis of proteins using polyacrylamide gel allow?
For the separation of proteins into their individual subunits.
What reagent and things will be used to denature the proteins before loading them into the gel?
1) SDS-strong anionic detergent sodium dodecyl sulfate
2) Reducing agents
What does the SDS do to the individual amino acids?
They bind to SDS and it coats them making them negatively charged.
What is the SDS-protein mass ratio?
What else does SDS block?
Disrupts hydrogen bonds, blocks hydrophobic interactions, and substantially unfolds the protein molecules thereby eliminating, or reducing at least, the difference in molecular form from secondary or tertiary structures.
What does the reducing agent dithiotrheitol (DTT) do?
Completely unfold proteins by reducing disulphide bonds.
Which move more slowly in the matrix, large proteins or small ones?
What does the separation ability of an SDS-PAGE gel depend on?
The concentration of polyacrylamide used to make the gel and the amount of cross linking.
What does the addition of bisacrylamide do?
Bifunctional agent that forms cross links of chains of polyacrylamide which adds rigidity and tensile strength to the gel forms pores which allow the SDS-protein complex to pass through.
What is the size of the pores dependent on?
The ratio of bisacrylamide:acrylamide.
What is the ratio of most gels?
1:29 which allows for the dissociation of peptides that differ in size by as little as 3%.
SDS will be carried out in what type of system?
A discontinuous buffer system in which the buffer in the reservoirs and the sample has a pH and ionic strength different than the buffer used to case the gel.
What is the buffer for the stacking gel?
0.5M Tris-HCl pH: 6.8
What is the buffer for the resolving gel?
1.5 M Tris-HCl pH: 8.82
What are the upper and lower buffer reservoirs?
Tris-glycine pH: 8.3
Which gel has higher porosity?
The stacking gel.
Where is the protein:SDS complex deposited after migrating through the stacking gel?
In a very thin zone (or stack) on the surface of the resolving gel.
What does the discontinuous buffer system effectively do?
Concentrates the protein into a very small volume allowing for increased resolution in the SDS -PAGE gel.
How are you able to determine the approximate size of the protein of interest?
By using molecular weight standards on the gel.
What is the Rf?
The ratio of the distance migrated by the molecule to that migrated by a marker dye-front.
Within the gel, where does a linear relationship exist?
Between the logarithm of the molecular weight of an SDS-denatured protein and its Rf.