BCH455.ProteinAnalysis

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Author:
cts0790
ID:
133683
Filename:
BCH455.ProteinAnalysis
Updated:
2012-02-09 00:27:55
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Test1
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Test1
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  1. Bradford Assay
    • Uses Coomassie G250dye which binds to basic aa (Lys, Arg, His) and aromatic side chains.
    • Reacts w. proteins > 3000Da
    • Measure color change at absorbance 595nm
    • Use BSA as standard

    • Advantages: easy and fast/ does not react with most buffers
    • Disadvantages: reacts w. detergents/ protein must be soluble at low pH/ variability twice Cu2+- based methods/ stains cuvettes/ reaction progresses with time, limiting # of consecutive samples.
  2. BCA Method
    Bicinchoninic Acid (BCA)

    • Advantages: Much more sensitive/ low protein-protein variability/ compatible with detergents/ simple to perform/ high repoducibility
    • Disadvantages: reacts with single amino acids (especially Cys, Trp, Tyr)/ reaction is continuous/ can't use chelating agents
  3. SDS PAGE
    • Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis
    • SDS interacts with the hydrophobic side chains and provides a negative charge so that the protein is linearized and overall negative charge --> separation now based on mass (not charge)
    • Gels are stained by Coomassie R250. The stain binds to insoluble protein
  4. SEC
    • Size Exclusion Chromatography
    • Gel matrix is particle based/molecular sieves (small particles with a network of pores of a given diameter that can accomodate molecules of a defined size
    • Small molecules enter pores quicker, large proeins migrate faster than sammer ones
    • MW of protein can only be determined if they share the same shape and properties of standards
  5. Ion Exchange Chromatography
    • separation based on charge
    • Charge on column opposite of desired protein
  6. Reversed-phase LC
    Separation of molecules is based upon their interaction with a hydrophobic matrix which is largely based on their polarity. Molecule are bound to the hydrophobic matrix in an aqueous polar buffer(stationary) and eluted from the matrix using a gradient of non-polar solvent(mobile).
  7. Affinity Chromatography
    • Separate protein by affinity inding specificity
    • Sample introduction/injection -->absorption of target molecules --> wash impurities --> elution of target molecules
    • Affinity resin components: biospecific ligand, support matrix, biochemically inert spacer
  8. Aromatic Content
    • Absorbance at 280nm
    • Trp > Tyr > Phe > S-S
    • Beer-Lambert Law: A = elc
    • determine concentration from absorbance value
  9. Peptide mass fingerprinting
    (sequencing)
    unknown protein is cleaved into smaller peptides. Mass spec is used to measure absolute masses of peptides and are compared to a database containing known protein sequences or even the genome.

    • Advantage: Time efficient and only masses of the peptides have to be known
    • Disadvantage: Protein sequence has to present in the
    • database of interest and not efficient for multiple proteins
  10. De novo peptide sequencing
    estimating a protein’s tertiary structure from its sequence alone
  11. Edman Degredation
    (sequencing)
    method of sequencing aa in a peppide by labeling then cleaving the amino-terminal residue without disrupting the peptide bonds between other amino acid residues

    • -PITC reacts w. the uncharged N-terminal gropu. The terminal amino acid is cleaved as a thiazolinone derivative then is selectively treated to form a PTH amino acid derivative that can be identified by using chromatography/electrophoresis.
    • -Only able to sequence 50 amino acids efficiently
    • -Alternatives to promote MS analysis via
    • protease cleavage
    • Trypsin (cleaves peptide bond at C-terminal end to R and K but not if next to P)
    • Endopeptidase V8 (peptide bond C-terminal to D, E)

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