Protein Techniques Flashcards
(27 cards)
How do you determine the molecular weight of proteins?
Multiply the number of amino acids by 110
What is the average MW of an amino acid?
110
Ion exchange chromatography separates based on what?
Electric charge
What determines charge in proteins?
pH
Affinity chromatography separates proteins based on what?
Binding specificity
How does affinity chromatography purify a specific protein?
By using a resin cross-linked with a specific ligand that tightly binds the protein, allowing all other proteins to be washed away
Molecular sieve chromatography separates proteins based on what?
Size
In molecular sieve chromatography, what size of molecules exits the column first? Why?
The larger molecules exit first because the smaller ones get stuck in the pores, and travel more slowly
Gel electrophoresis separates proteins based on what?
Size
How does gel electrophoresis work?
By using sodium dodecyl sulfate polyacrylamid gel, there is an overwhelming negative charge on the proteins. In an electrical field, proteins migrate toward the + pole (anode)
Isoelectric focusing separates proteins based on what?
Isoelectric point
In isoelectric focusing, proteins will migrate until they reach an area where their charge is what?
Zero - neutral
Which test separates proteins based on both isoelectric point and molecular weight?
2D gel electrophoresis
Which two tests are involved in 2D gel electrophoresis?
Isoelectric focusing
SDS-PAGE (gel electrophoresis)
Proteomics is used to identify differences in protein profile of what?
Two different cell populations
What does mass spectrometry measure?
Mass-to-charge ratio
What are antibodies?
Proteins produced by B lymphocytes that destroy foreign antigens
What are antigens?
Proteins (usually) that are recognized by other antibodies
What size must a protein be in order to undergo sequencing?
Fewer than 50 amino acids
Where does trypsin cleave?
On the carbonyl side of basic amino acids (Lys or Arg)
Where does chymotrypsin cleave?
Carbonyl side of aromatic (Phe or Tyr)
Where does S. aureus V8 protease cleave?
Carbonyl side of acidic amino acids (Glu or Asp)
Where does cyanogen bromide cleave?
Carbonyl side of methionine
In regards to protein sequencing, what is a fingerprint?
Regions of overlap resulting from protease fragmentation
