Lecture 9 Flashcards
Function of Polyacrylamide Gel Electrophoresis:
used for analytical separation of proteins when one doesn’t need a large amount of protein
Movement of molecules in PAGE:
- molecules will migrate through along an electric field through a gel matrix according to their size, shape and charge
- positive migrates to the cathode
- negative migrates to the anode
- buffers carry current for migration
Function of SDS-PAGE:
eliminates the effect of differences in shape, and so chain length (mass) is the sole determinant of the migration rate in SDS-gel electrophoresis
Function of SDS:
- detergent that denatures proteins, causing multimeric proteins to dissociate into their individual subunits
- coats the all polypeptide chains with a negative charge, therefore forcing the polypeptide chains into extended conformation with similar charge:mass ratios
Reagants that denature protein:
Urea, Guanidinium chloride, Beta-Mercaptoethanol, and SDS
Separation of SDS-Page:
separate only according to smaller proteins move more quickly towards the anode than large ones
Stained SDS-Page gel of proteins:
- Cell extract
- Affinity chromatography
- Ion exchange chromatography
- Size exclusion chromatography
- Marker lane known standards to determine size
Antibodies:
produced from B cells that bindi n a specific way to a protein, or an antigen
Separation of Western blotting:
separates protein by size and identifies by antibody
Process of Western blotting:
- run protein through an SDS-gel to separate by size
- transferred to nitrocellulose paper (gel matrix)
- proteins are transferred to the sheet via an electric current
- incubated w/ an antibody to bind to the protein of interest
- antibody is washed
- a secondary antibody is bound with an enzyme that produces color w/ light or color reaction where the primary and secondary antibody has bound
Separation of isoelectric focusing:
separates proteins by charge
Process of isoelectric focusing:
- buffer containing high or low pH is used to change the charge on a protein
- charge on a protein is determined by different amino acids it contains
- proteins migrate along gel until they reach a pH where their net charge is zero
2D-Gel Electrophoresis:
- isoelectric focusing gel separates by charge
- SDS-PAGE
Purpose of 2D-gel:
can be a valuable tool to compare proteins that are present in different tissues or in cancer tissue vs. normal tissue
Properties of enzymes:
- DO increase the rate of biochemical reactions
- DO NOT alter the equilibrium of a reaction (Keq)
- DO NOT affect the energetics of the reaction (Delta G)
- Binding is HIGHLY SPECIFIC, usually noncovalent interactions
ATP Hydrolysis:
aids in the energetics (net Delta G is negative)
Requirement of enzymes:
co-factors (enzymes or metals) for catalytic activity
Change in free energy (Delta G):
a valuable thermodynamic parameter to estimate if a reaction can take place and evaluate the energy cost of the reaction
Enzymes are generally:
proteins, and sometimes RNA (ribozymes)
Function of enzymes:
- acts as a catalyst for biochemical reactions
- increase the rate of biochemical reaction by lowering the activation energy to go from substrate to product
Specificity of enzymes:
EXTREMELY specific
1. substrate they recognize
2. reactions they catalyze
Example of an enzyme:
proteases catalyze the hydrolysis of peptide bonds in proteins
Rate:
velocity of reaction, the quantity of material that disappear or appear by unit of time
Rate units:
concentration per second or Ms ^-1
Equation of rate:
Rate Overtime:
Overtime, the rate of reaction decreases because the concentration of reactants decreases
Equation of rate in reversible reactions:
Equation of equilibrium constant via reaction rate:
Second order reactions:
enzyme-substrate reactions that are limited by the slowest step in the reaction, the rate-limiting step
Equation of second order reactions:
Transition State and Reaction Rate:
enzymes increase the rate of reactions by decreasing the activation energy barrier Delta G+ of the reaction
