Week 2 Flashcards
Where are water soluble, hydrophilic proteins found?
The aqueous compartments of the cell or body (cytosol, nucleoplasm, the lumen of organelles, blood, and extracellular secretions).
Where are water insoluble, hydrophobic proteins found?
In or on cellular membranes. Membrane proteins (integral or peripheral)
In the absence of detergent, how can cells be lysed?
Cells can be lysed by mechanical disruption (for example, using a Dounce homogenizer).
What is the result of centrifuging a cell lysate at high speed?
This will pellet all of the membranes and therefore all of the membrane proteins. The supernatant of the sample will contain most of the soluble proteins expressed by the cells.
What must be done to membrane proteins in order to purify them?
They must be extracted from the membrane with detergent.
What is SDS used for?
SDS is a type of detergent commonly used to extract integral membrane proteins from the membrane. It will denature proteins.
Triton
A mild detergent that will maintain the structural integrity of proteins and protein complexes.
Critical micelle concentration
Detergents will form micelles when their concentration rises above a specific threshold called the critical micelle concentration.
How do detergents (with the goal of lysing cells) work?
Cells can be lysed by adding detergent to them, which will disrupt membrane bilayers and extract proteins and lipids from the membranes. In a detergent lysate, most of the proteins in the cell will now by soluble.
In subcellular fractionation, what does the pellet contain after low-speed centrifugation?
- Whole cells
- Nuclei
- Cytoskeletons
In subcellular fractionation, what does the pellet contain after medium-speed centrifugation?
- Mitochondria
- Lysosomes
- Peroxisomes
In subcellular fractionation, what does the pellet contain after high-speed centrifugation?
- Microsomes
- Small vesicles
In subcellular fractionation, what does the supernatant contain after high-speed centrifugation?
The supernatant contains cytosol.
In subcellular fractionation, what does the pellet contain after very-high-speed centrifugation?
- Ribosomes
- Viruses
- Large macromolecules
How does column chromatography using gel filtration work?
This protein purification process separates proteins based on size. Bigger proteins come out first. Sample is applied to the top and solvent is continuously applied to the top of the column. Fractionated molecules are eluted and collected.
What types of assessments is column chromatography using gel filtration good for?
Good for assessing the mass of a native protein.
Tertiary and quaternary structure is maintained.
How does ion-exchange chromatography work?
Cation exchange or anion exchange columns (positive beads bind negatively charged proteins, negative beads bind positively charged proteins) Proteins are applied at a low NaCl concentration and are eluted by increasing the NaCl concentration of the buffer flowing through the column.
How do the beads of gel-filtration chromatography work?
Smaller particle get into the interior of the porous beads and therefore take longer to pass through the column than big proteins that go around the beads. The smaller the protein, the higher the probability it will flow into the bead and be retained for a longer time on the column.
How does affinity chromatography work?
Takes advantage of a specific binding interaction that your protein of interest may have. For example, some proteins bind cAMP (cyclic AMP) with high affinity. You can purchase sepharose beads with cAMP attached to load into a glass column. Proteins that bind cAMP will stick to the column while the majority of proteins will pass through. Free cAMP in solution can then be used to “elute” the column – meaning displace the bound protein so you can collect it from the solution flowing out of the column.
What does SDS-PAGE stand for?
SDS-polyacrylamide gel electrophoresis
How is the distance a protein migrates on a gel related to its mass?
The distance a protein migrates in the gel is inversely proportional to its mass.
What is the typical size of a protein?
A typical size of a protein is 50,000 Daltons (1 mole of this protein would weigh 50,000 grams).
How can the mass of an unknown protein be estimated using SDS-PAGE?
The mass of an unknown protein (in Daltons or kiloDaltons) can be estimated by comparing the distance it migrates in a gel to the migration of proteins with a known mass (standards).
In SDS-PAGE, what is the purpose of the reducing agent?
To break disulfide bonds.
What is the purpose of the SDS in the SDS-PAGE procedure?
To denature the polypeptide and coat it with negative charge.
When staining proteins in a gel with coomassie blue, what does the number of bands detected on a gel indicate?
Protein purity.
Column chromatography is an example of what type of method for studying proteins?
Protein purification
Enzyme assay (functional), SDS-PAGE, and Coomassie and silver stains are examples of what type of method for studying proteins?
Protein detection
Mass spectrometry is an example of what type of method for studying proteins?
Protein analysis
What is the purpose of x-ray crystallography?
Purified proteins can be crystallized and analyzed by x-ray crystallography to determine the three-dimensional structure of a protein.
What are cryo-electron microscopy and NMR used to determine?
Protein structure
What type of measurement is used to describe resolution?
Angstroms. A 1.2 angstrom structure is very high resolution and side chain details can be seen. 6 angstrom would be low resolution – general shape of the protein can be observed but typically one cannot resolve individual side chains
AlphaFold
A newly developed AI process that is very good at predicting the 3D structure of proteins from amino acid sequence.
What is time-of-flight mass spectrometry used to determine?
The mass of individual proteins (or derived peptides) in a simple protein mixture can be determined precisely using time-of-flight mass spec. (in short, used to identify an unknown protein in a sample)
