3-5 Purifying and Analyzing Proteins

Purifying Proteins
Concerns with purifying proteins…
o _______________ it from all other cell components and other similar proteins
o Methods are _______________ for each protein
o Want proteins to remain biologically _______________
o Must be done between _____°C and _____°C to avoid protein degradation and
Purification uses differences in…
o _______________
o _____ _______________
o __________
o _______________ _______________
Purification follows a series of general steps:
1. Prepare a solution of proteins from a cell.
2. Separate the mixture of proteins using ammonium sulfate as a _______________
agent, and then _______________ to isolate a fraction containing the desired protein.
3. Separation by _______________ works by washing the fraction containing the
desired protein through an insoluble material (matrix) using a specific solvent. This
divides the mixture into finer fractions, then those fractions must be assayed (tested)
for biological activity or other properties.
a. Chromatographic techniques include…
high-performance liquid chromatography (__________)
ii. ion-exchange chromatography
iii. gel-filtration chromatography, and
iv. affinity chromatography
* Read pages 68 and 69 to learn more about these different types
of chromatography.
Analytical Techniques
_______________ – separates proteins based on their migration in an electric field.
o Polyacrylamide gel electrophoresis (__________) uses a slightly basic gel matrix
with an electric field passed through it. The gel matrix retards the migration of
large molecules, thus the proteins are fractionated on the basis of
_______________ and _______________. The fractions of proteins can be
visualized by _______________.
o Sodium dodecyl suflate is sometimes added (__________) to overwhelm the
native charge on proteins so that the technique separates the proteins based on
__________ only. It can be used to assess the purity and the molecular weight of
the protein.
The “bands” of proteins can
be cut out of the gel and
isolated for structural analysis,
preparation of antibodies, or
other purposes.
__________ _______________ - determines the __________ of a molecule by
measuring the __________ it takes for a charged gas phase molecule to travel from the
injection point to a detector. The time depends on the __________and__________ of the
molecule. This technique was not applicable to biochemists until the late 1980s when
electrospray MS was developed to disperse charged protein molecules into a gaseous
stream of particles. Another new technique is matrix-assisted desorption ionization
(MALDI), which you can read about on page 71 of your book.
o This technique can determine the mass of a protein from _______________
quantities isolated from SDS-PAGE gel, and the correct mass can be determined
with an accuracy of less than the mass of a single _______________!
Mass Spectrometer Diagram