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