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- 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.
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
- 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
- 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
Ion Exchange Chromatography
- separation based on charge
- Charge on column opposite of desired protein
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).
- 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
- Absorbance at 280nm
- Trp > Tyr > Phe > S-S
- Beer-Lambert Law: A = elc
- determine concentration from absorbance value
Peptide mass fingerprinting
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
De novo peptide sequencing
estimating a protein’s tertiary structure from its sequence alone
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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