Chapter 3 - Exploring proteins Flashcards
Proteome
Is the functional representation of the genome.
Signifies a more complex level of information content
Peptide are used as
Hormones and pheromones
Neuropeptides
Antibiotics
Protection
Separation of proteins relies on
Charge, size, affinity for a ligand, hydrophobicity, thermal stability.
Differential configuration
Cells are disrupted in a homonizer and the homogenate is centrifuged. Used to purify protein from bacteria
Dialysis
The protein is placed inside the dialysis bag, made up of cellulose, which is then submerged in a buffer solution. This does not distinguish between proteins. Often used to dilute salt from solution
Gel filtration chromatography
Largest proteins come down first. Smaller proteins get into the porous beads made of agarose
Ion-exchange chromatography
Purity is achieved based on distinct molecular properties. Based on their net charge. If a protein has a net positive charge at ph = 7 it will usually bind to a column of beads containing carboxylate groups
Affinity chromatography
Can be used to isolate a protein. A protein mixture is passed through a column containing specific DNA sequences. Proteins with high affinity will bind. Then it’s washed with buffer to release unwanted proteins. Transcription factor is later released by adding a salt concentration solution
Electrophoresis
A molecule with a net charge will move in an electric field. They are forced to move through the same matrix
SDS page
Electrophoresis under denaturing conditions.
SDS is an an ionic detergent
B mercaptoethanol is added to reduce disulfide bonds
Complex of SDS
With a denatured protein, it has a large net negative charge.
The loaded buffer is added and boiled
Migration through the gel is directly proportional to the logarithm of mass.
Isoelectric focusing
Separated on the basis of their relative contents of acidic and basic residues. The isoelectric point of a protein is the pH at which its net charge is equal to zero
Isoelectric focusing pt. Two
Positive charge when phpl
How do you determine protein sequence?
Indirect: amino acid sequence
Direct: Edman degradation
Mass spectrometry
Edman degradation
The N-terminal amino acid of a polypeptide is labeled.
Tandem Mass Spect
Peptides can be fragmented by ions to generate a family of product ions. The carbonyl fragment of the cleaved peptide bond is ionized
Proteins can be cleaved
Done by specific chemical reagents. They protein backbone at particular amino acid residues in a predictable manner
Antibodies
Critical reagents for exploring the functions of proteins within the cell.
Vitro protein synthesis
Built from C TO N terminance
Chloride reacts with C group
How do you study protein structure?
X-ray chromatography
NMR
An antibody
Is itself a protei, it is synthesized by vertebrates in response to the presence of a foreign substance called an antigen
Immunoglobin
Consists of four chains, two heavy chains and red chains linked by disulfide bonds
Polyclonal
Are heterogeneous mixtures of antibodies, each specific one of the various epitopes of an antigen
Monoclonal
All identical, produced by clones of a single antibody-producing cell
Preparation of monoclonal antibodies
Think of the rat. Hybridoma cells
How to make polyclonal
Purify protein you want the antibody to grow in, inject protein in to animal, wait a few weeks while animals create antibodies for the antigen, collect blood
Antibody techniques
ELISA
Western Blotting
Antibodies can be used as exquisitely specific analytic reagents to quantify the amount of a protein or other antigen present
Indirect Elisa
Starts with an antigen coated well
Sandwich Elisa
Starts with monoclonal antibody coated well
Western blotting
To quantify very small quantities of proteins. The subject is subjected to electrophoresis on a SDS polyacrylamide gel. A primary and secondary antibody is added
