CHMI-2227EL Lecture Notes - Lecture 7: Biuret, Tryptophan, Phenylalanine
27 Jun 2018
School
Course
Professor
Biochemistry - Day 7 2018.02.02
Purification of Proteins Techniques Continued
-Very powerful method but:
-Media is very expensive, therefore we cannot perform large scale purifications
-Limited availability of ligand-bonded beads
-Requires prior knowledge of a ligand that can bind to the protein of interest
Electrophoresis
-It separated protein molecules on the basis
of their charge and size
-Proteins are allowed to migrate in a gel
matrix (polyacrylamide or agarose) under the
influence of an electric field
-After electrophoresis, the gel is stained with
a dye that binds to proteins or by
electroblotting the proteins into nitrocellulose membrane and probing with antibody
that is specific for the fired proteins
-This immunoblotting technique is called western blotting
SDS polyacrylamide gel elecrophoresis (SDS-PAGE for
short)
-SDS is a negatively charged detergent use to denature
proteins
-SDS disrupts the native conformation of proteins and
place them all in a linear shape
-This will usually be used in combination with other
reagents like beta-mercaptoethanol COPY REST
Ultracentrifugation
-The Svedberg developed the ultracentrifuge in 1923
-This instrument can generate centrifugal forces over 600 000 G’s and has been used
to determine the molecular weight and subunit composition of proteins
-The rate of sedimentation of proteins depends on their shape and their molecular
weight
Enzyme-linked immunosorbent assay (ELISA)
1. We fix an antibody on solid support (usually a 96-well polystyrene plate)
2. Then we incubate our protein sample on the antibody coated polystyrene surface
(the desired protein will bind the antibody)
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Media is very expensive, therefore we cannot perform large scale puri cations. Requires prior knowledge of a ligand that can bind to the protein of interest. It separated protein molecules on the basis of their charge and size. Proteins are allowed to migrate in a gel matrix (polyacrylamide or agarose) under the in uence of an electric eld. After electrophoresis, the gel is stained with a dye that binds to proteins or by electroblotting the proteins into nitrocellulose membrane and probing with antibody that is speci c for the red proteins. This immunoblotting technique is called western blotting. Sds is a negatively charged detergent use to denature proteins. Sds disrupts the native conformation of proteins and place them all in a linear shape. This will usually be used in combination with other reagents like beta-mercaptoethanol copy rest. The svedberg developed the ultracentrifuge in 1923.
