day 2: protein techniques + enzymes Flashcards
proteome
like a genome, but for proteins (Proteome: function of all proteins)
proteins can be purified based on:
net charge
size
sedementation rate
solubility
binding capabilities
how do you liberate materials from a cell? (to find proteins of interest)
sonication or homogenation
once you’ve liberated material from cells what do you want to do?
separate out the proteins from each other to find protein of interest
how do you separate proteins?
9 techniques
how does separating proteins work
Separation methods target the differences in protein structures such as charge, size, solubility, and specificity of binding sites or active sites. The “fractions” are collected and assayed
9 techniques for separating proteins
- Salting out
- Gel filtration chromatography
- Ion exchange chromatography
- Affinity chromatography
- Polyacrylamide Gel Electrophoresis (PAGE)
- SDS
- Isoelectric focusing
- Density gradient centrifugation
- immunoprecipitation
the 3 steps for getting to your identified protein
- Release all proteins from the cells
- Separate types of proteins from each other (fractionation)
- Identify which fraction contains the protein of interest (assay)
identification assays
- Enzyme activity if the protein is an enzyme (e.g., colorimetric assay)
- Bioassays if the protein has a known effect on a tissue (e.g. slows heart rate)
- Antibody (Ab) binding with an antibody generated in response to that protein (note: Ab is covalently attached to a fluorescent or radioactive molecule for detection)
- Ligand binding if the specificity of the protein’s binding site is known (e.g. the lectin, Con A binds glucose or estrogen receptor binds estrogen. The ligand is made radioactive for detection)
how would you go about characterizing the purified proteins?
The protein can be digested into amino acids to determine the composition (the percent of each different amino acid in the protein)
- The sequence of amino acids can be determine using stepwise removal of each amino acid by Edman degradation or by using tandem mass spectroscopy
- Xray crystallography shows 3-D shape
- 2-D NMR techniques allow some features of 3-D
How to break open cells:
1) You’d literally get a beef liver, put it in a blender. Keep it cold, blend extremely short time- got ‘crude extract’ aka starting material
2) Might also use the homogenizer tube; glass tube, mortar + pestle style, smushes cell membranes down
3) Last way: sonicator. Uses sound waves.
Bcaterial cells super hard to open up – use a french press. Plant cells too
Differential centrifugation
Cells are disrupted in a homogenizer and the resulting mixture, called the homogenate, is centrifuged in a step-by-step fashion of increasing centrifugal force. The denser material will form a pellet at lower centrifugal force than will the less-dense material.
Most dense is nuclear stuff. Next most dense is mitochrondria. Then microsomal. Then cytosol.
Cytosol:
all soluble components of cell that are not particulate. Aka all dissolved proteins, free ribosomes, all soluble portion of cytoplasm.
how do u remove salt after salting out
dialysis
Gel filtration chromatography
•separates protein according to size- called gel filtration. The big protein cant fit in the little pore– goes straight down. Intermediate protein can fit in some but not all pores, some sinks down, some stays. Little guy fits right into the pores of the beads, stays on top. Little proteins exit the column last because they can hang out in the pores of the beads.
Ion exchange chromatography
separation of proteins on the basis of charge. The beads in the column have a + or - charge
Affinity chromatography
takes advantage of the fact that some proteins have a high affinity for specific chemicals or chemical groups. Beads are made with the specific chemical attached. A protein mixture is passed through the column. Only protein with affinity for the attached group will be retained.
native gels vs denaturing gels
Proteins will migrate in an electrical field because they are charged. When the migration occurs in a gel, the process is called gel electrophoresis. This can either a “denaturing gel” with SDS as the denaturant or a “native gel”.
Native gels separate by charge and mass and preserve the functional protein shapes.
denaturing migrate based on SIZE ONLY
SDS separates proteins based on:
size
Isoelectric focusing
allows separation of proteins in a gel on the basis of their relative amounts of acidic and basic amino acids. If a mixture of proteins is placed in a gel with a pH gradient and an electrical field is applied, proteins will migrate to their isoelectric point, the pH at which they have no net charge.
Density Gradient Centrifugation
Ultracentrifugation can be used to examine proteins. When subjected to a centrifugal force, the rate of movement of the particle is defined by the sedimentation coefficient, s. When centrifugation is through increasing densities it is called Density Gradient Centrifugation
its a way of separating out proteins from each other- one of the methods of fractionation
Identification assays enable scientists to:
locate which fraction has the protein of interest using assays for activity or binding ability
Immunological techniques can be used to both purify and identify proteins
The estrogen receptor binds the steroid hormone estradiol tightly and with great specificity.
The estrogen receptor has no enzymatic activity, but can be purified by immunological techniques and the use of gradient centrifugation
can you generate antibodies 2 specific proteins?
yes
