Lecture 14 & 15 Flashcards
Preparative & Analytical Technique
Why are proteins purified?
So the structure and function of them can be studied in isolation (without any other materials interfering)
How are proteins purified?
Breaking up the cells, then filtering to find the proteins by differential centrifugation, then by preparative techniques like
1) Salting out
2) Column Chromatography
What are the 3 ways cells can be broken up/lysed?
1) Sonication
2) Shearing
3) Mild detergents
Explain sonication
a metal rod is placed into a vial and vibrations creates a build up of pressure until the cells burst
Explain shearing
Rather cells are squished into a tiny hole or squished against the wall
Explain Mild detergents
They lyse the cell by dissolving their hydrophobic layers
What is centrifugation?
when materials are spun so hard that gravity sorts it out by density
Differential Centrifugation
Used to filter the proteins from lyse cells. Each time the heaviest component goes to the bottom and the supernatant (liquid with the lighter materials) is placed into a separate vial to be centrifuged again. this process is repeated until the protein wanted is the heaviest component
Explain salting out
Used to find specific protein within many. The proteins are lest soluble when neutral and in a high salt concentrations. They precipitate out of the solution by clumping up at their isoelectric point. Ammonium sulphate is the most common solution used.
what is the difference between analytical and preparative techniques?
preparative techniques are used to filter to find a specific protein. analytical techniques are used to find the structure and properties
What is Column Chromatography?
When proteins get filtered based on their physical and chemical properties
What are the three types of column chromatography?
1) Ion exchange chromatography
2) Gel (size chromatography)
3) Affinity Chromatography
What is ion exchange?
A preparative technique. Protein get separated by charge. Depending on the type of resin, proteins get electrostatically bonded, while others fall through. To obtain the bonded protein, the pH of the solution is changed.
What is Gel (size exclusion) chromatography?
A preparative technique. mixture is pumped into the gel matrix that contains many small holes. the smaller protein will get stuck in the holes and take longer to leave while the bigger ones will exit quicker
What is affinity chromatography?
A preparative technique. The specific protein binds to the ligand that is covalently bonded to resin. All the other proteins easily washed through as they don’t bond to anything. Then, to release the protein, the solution is pumped both ligands so the protein will bind onto those free ligands and filter through.
what is SDS-Page Gel Electrophoresis?
It separates protein by mass. The proteins are surrounded by negatively charged SDS molecules. The top is the gel is negatively charged and the bottom is positively charged. Both are covered with a buffer. The smaller proteins are able to move towards the positive side because the gel is criss crossed so the big ones cans move down.
How is molecular weight it estimated?
The larger the portion, the less it moves. The retention factor is the distance migrated by the protein divided by distance go the dye front. (a lot like (TLC).
What is a protein ladder?
a markings of the movement of proteins with known molecular weight. used to estimated the weight of unknown proteins
what is a 2D electrophoresis?
it uses the isoelectric focusing (overall charge of protein) first then by molecular mass. once they reach their isoelectric point, they become neutral and they don’t move.
Why is 2D electrophoresis a good method to separate for MS analysis?
You can analyze multiple proteins at a time and figure out it’s properties
What is a proteome?
entire set of proteins from an organism. Secretary protein set= secretome.
NMR?
Nuclear Magnetic Resonance shows the direction the atoms spin.
Limits
- limited by size (small only)
- sensitive to local environment
X-ray crystallography
exposes the crystals of protein by removing water.
Pros
- more detailed than NMR
- no size limit
Limit
- open to human error
- picture is static, not using for a protein you want to change
Antibodies
a protein, also called immunoglobins made by the body in response to a forge in substance, antigen.
Parts of a antibody
shaped like a Y, there 4 chains. 2 heavy chain that make the 2 halves of the Y. 2 light chains outline the heads of the Y. 2 Fab domains and one Fc domain.
Fab domain
antigen binding domain
Fc domain
binding to the receptors of the host cell (crystallizable fragment)
Epitope
a specific spot on a antigen that the antibody binds to. It binds due to stereochemical complementary.
What is ELIZA?
Enzyme-Linked Immunosorbent Assay- it detects proteins and quantification
How does ELIZA work?
1) the well is coated by antigens
2) antibodies that specifically bind to the antigens are placed in the well and bind to the antigens
3) then enzyme looked antibodies bind to the primary antibodies that are bound to the antigen
4) those enzymes will release a colour as a indicator when bonded to a substrate the is put in the well
5) the brightness of the colour quantifies the amount of antibodies present
What is Immunoblotting (westen blotting)?
for of proteins detection and quantification
How does Immunoglobulin (western blotting) work?
1) First run a SDS electrophoresis
2) Transfer to the filter membrane
3) wash with your primary antibody
4) wash with ur secondary antibody
5) add substrate and it’ll admit colour you want
How does enzyme linked- secondary antibody bind to primary antibody?
The enzyme linked secondary antibody binds to the Fc domain of the primary antibody which is the same of every type of antibody and does not change.
