lecture 8 - quantifying purity Flashcards
spectophotometry
- absorbance (A) = log (Io/I)
- Io = initial light passed through sample
- I = light transmitted and detected after passing through the sample
Beer-Lambert Law
- A = Ecl
- E = molar coefficient (L/mol/cm) - unique for each molecular amino acid - how much light can be absorbed at specific wavelength
- c= concentration (mol/L)
- l= path length (usually 1cm)
- Trp > Tyr > Phe - only ones that absorb UV
B-L is useful for determining the concentrations of what proteins?
pure proteins only
B-L is not useful for:
determining concentrations in a mixture of proteins
B-L: for a crude mixture of proteins with a typical or average amino acid concentration, absorbance is equal to:
-A280 = [total protein] in mg/mL
-[P] = 1 mg/mL then A280 = 1
(just approximate and only for mixtures of many proteins)
what is a method for measuring total protein?
Bradford assay
how does a bradford assay work? is it dependent on amino acid composition?
- uses coomassie die which binds protein and absorbs 600nm
- more sensitive
- not dependent on amino acid composition
- use a standard curve to measure total protein (look at absorbance on y axis then follow to mg/mL on x axis)
what assays are useful for the specific measurement of target protein?
- immunoassay
- enzyme assay
what does an immunoassay detect?
antibodies that bind to the target protein
what does an enzyme assay measure?
the reaction catalyzed by target protein
- if Sāā>P by a target enzyme
- then can measure increase [S] or [P] using spectophotometry
what is an enzyme unit?
1 unit = amount of enzyme that catalyzes the conversion of 1umol of S in 1 min at 25 degrees C
specific activity equation
units/ total protein in mg
what happens to specific enzyme activity as purity increases?
specific activity of a protein increases up to a maximum point where the protein is pure
what does # units measure? (purity or yeild)
yeild
what does specific activity measure? (purity or yeild)
purity
