2.1 - Protein Techniques
35 Cards in this Set
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What methods can you use to detect proteins and amino acids?
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Colormetric - use dyes like coomassie blue
UV - 280 nm
Assays
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What are the two protein assays? Describe.
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Enzyme assay - rate of product formation proportional to amount of enzyme present
Immunoassay - use antibodies to detect proteins
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What are the two immunoassays?
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Radioimmunoassay
Enzyme Linked Immunosorbent Assay (ELISA)
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What is the difference activity and specific activity?
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Activity is total protein
Specific Activity is target protein
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What protein characteristic does salting out target?
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Solubility
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What protein characteristic does ion exchange, electropheresis, and isoelectric focusing target?
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Charge
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What protein characteristic does hydrophobic interaction chromatography target?
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Hydrophobic interactions
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What protein characteristic does gel filtration and SDS-PAGE target?
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Size
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What protein characteristic does affinity chromatography target?
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Binding specificity
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What is the process of salting out?
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Add salt and precipitate will form which can then be precipitated out.
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The (smaller, larger) the pKa, the less soluble the protein. It will, thus, precipitate out quicker the protein.
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Smaller
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When do you use salting out?
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If the Ksp's are far apart
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What is the process of an ion exchange?
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Nonpolar matrix with attached charged particles
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Which fragments will elute first?
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If it is a cation exchange:
(-) repulsed, come out first
then (0)
(+) attracted so does last
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What is the process of affinity chromatography?
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**** binds
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How do you unstick affinity binding?
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Change charge
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What is the process of gel filtration?
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Largest elutes first because it bypasses everything.
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In electropheresis, the (smallest/largest) goes furthest.
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Smallest
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In 2D electropheresis, fragment are sorted by?
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Size and charge due to a pH gradient in the gel
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Where does Trypsin cut?
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After Lys and Arg
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Where does Chymotrypsin cut?
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After aromatic amino acids
Phe, Tyr, Trp
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Where does CNBr cut?
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After Met
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Where does Endopeptidase V8 cut?
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After Glu
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Where does Elastase cut?
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After small residues
Gly, Ala, Ser, Val
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What does isoelectric focusing do?
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Pinpoints the pI
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What is the process of ultracentrifugation?
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Separates slow vs. fast sedimenting precipitates
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What is Edmund Degradation?
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Method of choice for protein sequencing
Removes one amino acid at a time from the N-terminus
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What is the limit for Edmund Degradation?
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About 50 residues
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What are the downsides to Edmund Degradation?
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Can only sequence 50 residues at a time
Problems with disulfide bonds
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Amino acid composition is analyzed by?
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HPLC
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What do you use for N-terminus determination?
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FDNB or Dansyl Chloride
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What do you use for C-terminus determination?
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Carboxypeptidase
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Where does Pepsin cleave?
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Cleaves after Leu, Phe, Trp, Tyr
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Endopeptidase
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Enzymes of bacterial origin that cleaves peptide bonds within a protein chain at specific sites
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What is the Edmuns reagent?
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phenylisothicyanate
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Signal Transduction - Flashcards