Mirco Lab Midterm: Lecture 6 Flashcards
1
Q
Overview of the current methods of protein quantitation in solution
A
Protein amino acid analysis. This is the most
accurate method for protein quantification,
but requires sophisticated instruments
- UV spectrometry
- Colorimetric methods
- Mass spectrometry
2
Q
Describe UV spectrometry
A
Quantifies the concentration of purified proteins by
measuring the absorbance of aromatic amino acids
tryptophan, tyrosine and cysteine at 280nm
- Requires pure protein, incompatible with
contaminants like nucleic acids that have similar
absorption spectrum, absorption coefficient must be
known - Simple, requires very small sample volume
- Does not need standard curve when the molar
absorption coefficient is known
3
Q
What are colorimetric assays
A
- Determine the concentration of individual
proteins or mixtures of proteins in solution - Dye-binding: Lowry, BCA (bicinchoninic acid),
Bradford - Fluorescent methods
- Sensitive to solvents: salts, detergents
- The concentration of the protein in a sample is
determined by a standard curve of a reference
standard protein solution such as BSA
4
Q
Proteins for standard curves
A
Pure proteins
- Known concentration measured by an
independent method - Broad linear dynamic range
- Fitting linear or nonlinear regression models
to the quantitative data
5
Q
What are the principles of standard curve assays?
A
- Identically treated samples are directly comparable,
because the protein quantity is the only cause for
difference in final absorbance - if an unknown sample
and a standard have the same absorbance then they
contain the same concentration of protein - The concentration of an unknown sample determined by
a standard curve is expressed in the same units as the
standards used to make the standard curve (i.e. mg/ml) - If the sample has been diluted the dilution factor is
considered after the measurement
6
Q
Look at slide for graphs
A
