Lecture 8 Protein Purity and Identification Flashcards
what method assesses protein purity?
SDS-PAGE
What makes a protein unique?
A protein’s primary structure
What are the different methods to identify proteins? IME
Immunoblotting, Mass Spectrometry,
Edman Degradation
What are the steps of Western Blotting? (5) STBAD
SDS-PAGE
Transfer to a polymer sheet
Blocking non-specific sites
Antibody Binding
Detection of bound antibodies
How does the blocking solution prevent non-specific binding?
by coating the membrane.
What is the purpose of the blocking solution in Western Blotting?
prevents non-specific binding
What does the primary antibody do in Western Blotting?
binds specifically to the protein of interest.
How is the signal amplified in Western Blotting?
A secondary antibody binds to the primary antibody
What are examples of post-translational modifications detected by Western Blot? PUM
Phosphorylation, Ubiquitinylation,
Methylation
Describe the process of Ubiquitinylation.
Adds ubiquitin, marks for degradation
How does mass spectrometry identify proteins?
It ionizes proteins and separates them based on their atomic weights.
What is the biggest limitation of immunoblotting techniques?
availability of good antibodies for the protein of interest.
What factors affect the time of flight in mass spectrometry and how? (how do small, large and positively charged fragments move)
Smaller fragments move faster
Larger fragments move slower
More positively charged fragments move faster
how does Edman Degradation work?
removing one amino acid at a time.
What is Edman Degradation used for?
It is used to sequence proteins
