Proteins Flashcards
What functions do proteins do?
Enzyme catalysis Transport and storage Immune protection Mechanised and storage Control of growth and differentiation
What is the purpose of electrophoresis?
To separate proteins according to molecular weight
What do you need to do to get electrophoresis to work?
You need to make proteins the same shape and charge. To do this you need to denature and and unfold them with SDS.
Sodium Dodecyl Sulphate
How does sodium dodecyl sulphate work?
It is a detergent that dissolves hydrophobic molecules and has a negative charge. It binds proteins constantly (1g of protein for 1.4g of SDS)
It masks the net charge of the protein and unfolds it. Therefor no secondary, tertiary or quaternary structures.
What are reducing agents?
They are usually attached to break disulphide bonds (detaching subunits to ensure separation)
How does polyacrylamide gel work?
It puts the proteins in an environment that allows different size proteins to move at different rates based on MW.
It is a gel formed of polymers of acrylamide.
An electric current pulls the proteins through the gel which is made of a labyrinth of tunnels through meshwork of fibres.
How does electrophoresis work?
The proteins enter the gel at the same time. They all have the same force per mass pulling them towards the same end.
Smaller proteins move faster then larger ones so they separate according to molecular weight.
What is the structure of discontinuous gel electrophoresis?
Two different gels (one on top of the other) with different pH and acrylamide concentrations.
The first is the stacking gel, second is resolving gel.
Stacking: 4% acrylamide
Resolving: 10-15%
How does discontinuous gel electrophoresis work?
All the molecules slow down when they enter the stacking gel. It concentrates the proteins in a narrow band between the stacking and resolving gels.
The proteins don’t separate until they enter the resolving gel.
What are the features of gradient gel electrophoresis?
The proteins migrate through an increasing percentage of acrylamide (e.g. 4-20%h
The gel then becomes increasingly restrictive for the proteins to migrate.
How to visualise the proteins?
Once the gel is run, to visualise the movement of proteins you first need to fix them in weak acid so they don’t move. Then you need to stain them with commasie blue dye and then remove excess dye.
How to determine Molecular weight?
You need to run a lane with proteins of a known molecular weight.
A linear relationship exists between the logarithm of molecular weight of a denatures polypeptide and it’s rF.
Log(MW) vs rF on standard curve
How to calculate rF?
Relative mobility
Distance migrated by the band divided by the distance migrated by the dye front
Western blot or protein immunoblot?
This is a technique for identifying a particular protein using antibodies after electrophoretic separation in a gel and transfer to a membrane.
Question: is a particular protein present in the same?
- Separate the proteins using SDS page
- Place the nitrocellulose membrane on the gel and using electrophoresis, drive the protein bands onto the membrane.
The proteins are now on the membrane surface.
3.Incubate the membrane with an antibody for the protein of interest. - Then incubate with a secondary antibody against the first. The secondary antibody is attached to an enzyme and will bind to the primary.
- Incubate with the substrate for the enzyme to give a fluorescent products.
- Visible band on membrane = specific protein.
