Lecture 2: Extracting Proteins Flashcards
Why is protein extraction important?
After sourcing our protein, it is important to extract it properly. We need to know how to detect the protein, break cells, centrifuge, place it in buffer and purify by precipitation.
At all stages of the assay we need to test the presence and purity of a protein. It is also good to test the activity of a protein.
How can we detect protein concentration?
The dye Coomassie Brilliant Blue G250 is used.
• It is green-brown in acidic solution, but it becomes blue when bound to protein.
• We can then use absorbance to measure concentration.
• The dye binds to a number of side chains (aromatics, arginine and histidine).
We can just use the protein without the dye to find concentration. Many proteins absorb at 280nm due to trp, tyr, phe and cys. An extinction coefficient can be estimated from the sequence.
How is SDS-PAGE used?
Sodium dodecylsulphate polyacrylamide gel electrophoresis is a method for visualising proteins.
• The protein is heated with SDS which binds to the hydrophobic parts of the protein, unfolds it and gives it a negative charge.
• A current is then applied across the gel to draw the SDS coated protein through the gel. We can estimate protein size through the rate of movement.
• We can use Coomassie to show all proteins.
How does Western blotting work?
Western blotting is a method to show the presence of a specific protein.
• The proteins are moved from a gel to a membrane.
• The membrane is blocked using milk proteins.
• The protein of interest is detected with an antibody and a secondary antibody linked to a fluorophore, horse radish peroxidase etc.
Why do we use functional assays?
We want to know if our protein is still functioning.
• We can test binding or catalysis.
• We can use special ligands as labels.
• For example, charybdotoxin (CTX) binds to many potassium channels if they are correctly folded.
• Radiolabelled CTX is incubated with the protein in the presence of different concentrations of unlabelled CTX.
• The samples are washed on filter papers using a vacuum manifold before the radioactivity is counted.
• This gives binding affinities and reports on the state of the channel protein.
• Enzyme assays look for loss of substrate or gain of product using an easily detected molecule. A classic example is the cleavage of ONPG by beta-galactosidase. When cleaved, the ONP product is yellow (420 nm detection).
What do we add to the resuspension solution?
- Buffers: Buffers are used to control pH to prevent denaturation. We choose a buffer which has a pKa close to where we are working and one that is useful at the pH where the protein is normally found. Buffers generally work within 1 pH unit of the pKa.
- Salt: Proteins have evolved to fold in a salt solution of 150mM NaCl, not just pure water.
- Reducing agents: The inside of a cell is a reducing environment. For extracellular proteins we leave them out, so DS bonds can form. We normally use DTT and beta-mercaptoethanol.
- Ligands: Some proteins require the presence of ligands, co-factors or prosthetic groups.
- Protease inhibitors: They come in a tablet with multiple inhibitors. PMSF: Inhibits serine proteases.
- Detergents: They are essential for making membrane proteins. They can also be used when a protein has an exposed hydrophobic patch.
- Sucrose/glycerol: Sucrose or glycerol can increase the density of a solution. This can help maintain the folded state. Glycerol interacts with small hydrophobic patches, so it prevents aggregation, it can also prevent freezing.
How do we disrupt the cell membrane?
If the protein is secreted, we can purify it from the media in which the cells were grown. However, we need to break the cells if the protein is inside. Mammalian cells are easier to break than E. coli or yeast.
Gentler methods: For mammalian/insect cells.
• Osmotic lysis: We place the cell in a buffer, so water will flow inside and burst them.
• Dounce homogenisation: A plunger within a tube. They fit very tightly so the cells are forced apart. (Used for mashing up salmon testicles in first year).
• Blade homogeniser: Blender.
• Squeezing through a bent needle.
Harsher methods: Used for E. coli/yeast.
• Cell disrupter: Forces the cells through a small hole at high pressure. The shear forces lead to cell lysis.
• Sonicator: High pressure sound waves generate shear forces and cause cavitation (bubbles). We need to be careful though, as it can cause the cell suspension to warm up.
How does centrifugation work?
- The denser and more massive a molecule is, the faster it will sediment in a centrifugal field. Shape is important too, a spherical molecule will sediment faster than a flat molecule.
- With E. coli we use a low spin speed to go from a suspension to a lysate. We then use a higher speed, longer spin to clarify the broken cells.
- We can also use differential centrifugation to separate out different cell components. We can perform a series of centrifugations at different speeds.
How can we use ammonium sulphate precipitation?
When we have a soluble extract containing the protein. We salt out the protein.
• The salt reduces the amount of solvent available to interact with the protein.
• Different proteins will precipitate at different concentrations.
• We need to run trials to find what conc we need.
