Electrophoresis Flashcards
(17 cards)
What’s the function of SDS in protien electrophoresis
To bind to exposed hydrophobic sites on the protein, coating it in negative charges. Stopping it from sticking together, and keeping the protein soluble
What happens if a protien is heat-denatured in the absence of SDS
Hydrophic sites will be exposed and stick together.
Clumps of protein will be insoluble
Why is dye used in the electrophoresis process
For easy loading of samples and easy following of their progress
Why do you need to avoid the formation of secondary structures in molecules you want to analyse by electrophoresis
As this can change the shape of the molecules, so the molecular size can be predicted by its profile
What is the function of reducing substances
They bread disulphide bonds between cystine residue
So separate protein complexes held by these bonds
Why are RNA molecules run at high temperatures in agarose gels containing formamide
RNA is prone to forming intramolcular base pairs that will make secondary structures.
Formamide breaks these structures
What is the charge of SDS
Negatively charged
What’s the difference between denaturing and native gel electrophoresis
In denaturing - the molecule, protien, will be denatured
In native - the molecule remains in its native state
Which chemical compound is usually used in gel electrophoresis to keep denatured protiens in a soluble state
SDS - sodium dodecyl sulphate
What is the basis for the separation of molecules through electrophoresis
Uniformly charged molecules will be separated on the basis of their size
Small molecules move faster
What happens if you add SDS to a protien but dont heat denature the protien
The SDS may denature the protien by breaking some of its intermolecular bonds
But sometimes the SDS is not enough to do this so the protien will not denature
A protein complex is made up of two subunits which are held together by a disulphide bond – one subunit has a molecular mass of 50 kDa and the other one has a molecular mass of 25 kDa. You run this complex on a denaturing SDS gel in the presence of reducing substances. How many bands will you detect?
In the presence of a reducing agent , disulphide bonds will be broken
2 bands
What should you NOT do when running a native protien gel
Avoid any steps leading to denaturation
Eg heat, SDS or reducing agents
Which way do protein molecules move in an electric field at ph7
Depending on the charge of the protien,
It moves to the anode if negatively charged
It doesn’t move at all, if neutral
It move towards the cathode , if positively charged
What is the driving force in any kind of electrophoresis
The existence of an electric field that allows molecules to move and be separated
Why are buffers used in electrophoresis
To avoid any changes in the pH during the electrophoresis that may affect the charge of the analysed molecule
What molecules are usually analysed by SDS-PAGE
Proteins
