Polyacrylamide gel electrophoresis (PAGE) of proteins Flashcards
What is electrophoresis?
movement of charged molecules in response to an electric field resulting into their separation
In free solution, what is mobility dependent on?
Mainly on net charge
When gel is present, what is mobility dependent on?
Size and shape of protein
What is the relationship between charge and mobility?
Higher the charge, higher the mobility
For electrophoresis, where do negative charges attracted towards? Positive charges?
Negative charge moves towards the anode (+)
Positive charge moves toward the cathode (-)
What is polyacrylamide gel?
Cross linked polymers of acrylamide and bis-acrylamide
What are the size of the pores in polyacrylamide gel dependent on?
total concentration (%T, w/v) of acrylamide and bis-acrylamide and % Cross linkers (w/w)
Usually the separating gel % varies between 4-20%
What is electrophoretic mobility dependent on?
Charge, molecular weight, charge to mass ratio, gel concentration and viscosity
Higher electric field, higher mobility
What is the Ferguson plot?
a relationship between the concentration of gel and the electrophoretic mobility of a protein
Same charge but different mass
All proteins extrapolate the same mobility at 0% gel
What are the two types of PAGE?
Native-PAGE (non-denaturing)
SDS-PAGE (denaturing)
What is native-PAGE?
Separation based on native charge, size, and shape
What is SDS-PAGE?
Separation is purely based on size
SDS will make proteins negative so size is the only variability (native charged masked)
SDS denatures the protein
How does SDS denature the protein?
Dissociates subunits
Unfolds polypeptide chains (linear)
Binds to denatured protein and imparts negative charge to the polypeptide chain
If a native-PAGE shows a band high in the gel and then in SDS the band goes down the gel, what does this mean?
Shows that this is made up of 2 subunits SO
Protein is most likely a homodimer with non-covalent bonds
What do reducing agents do?
break down the disulfide bonds
B-mercaptoethanol (2-ME) and Dithiothreitol (DTT)
What is the relationship between mobility and gel concentration?
Mobility inversely proportional to gel concentration (Ferguson chart)
Bigger size, lower mobility
How is the separation pattern of proteins different under non-reduced and reduced conditions of SDS-PAGE?
In non-reduced conditions, disulfide bond does not break, stay as a dimer, and the molecular weight stays the same as native
In reduced conditions, disulfide bond gets cleaved and now are monomers which makes two subunits of half the native weight. It also goes down the gel.
How to get molecular weight of protein?
Using Rf
Rf = distance migrated by the protein / distance migrated by the dye front
Values range from 0-1
