Term
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Definition
The pH at which the average NET charge of a molecule is zero.
For an uncharged Amino Acid, take the average of the two pKa's
Amino Acid w/ titratable R-group, the average of the two pKa's surrounding the species with total charge of zero |
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Term
| 5 Steps to protein purification |
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Definition
Rupture cells (freeze, sonicate, french press, vortex)
Collect proper fraction
Purify protein of interest by selecting for characteristics of that protein (will decrease yield)
Determine purity of protein. If not pure, repeat previous steps.
Characterize purified protein |
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Term
Protein purification & seperation:
How are proteins different from each other? |
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Definition
Solubility: (NH4)2SO4 goes SLOWLY, aka "salting out"
Charge @ a given pH (pI)
Size
Binding to a particular substance/material |
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Term
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Definition
Dialysis (removing salt & small molecules, not proteins)
Centricons (no big molecules, do it 3 times, similar to dialysis)
Column Chromatography (Ion exchange, Size exclusion - smaller takes longer, affinity - beads/tags to catch His) |
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Term
Total Protein ?
Yield?
Activity? |
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Definition
T. protein : the total, including what you want to get rid of.
Yield: your protein of interest only.
Activity: measures "your" enzyme |
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Term
Total Activity?
Specific Activity? |
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Definition
Total Activity: what you really want out of the protein
Specific Activity: purity of protein;
= (T activity / T protein) |
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Term
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Definition
Primary (actual sequence)
Secondary (conformations incl alpha-helix & Beta sheet)
Tertiary (complete 3D picture of single polypeptide chain)
Quaternary (not always done; arrangement of individual subunits into the complete protein) |
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Term
| Enzymes are _________ of amino acids |
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Definition
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Term
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Definition
Peptide bond occurs through a condensation reaction (H2O is released)
Has N & C - terminus. Always name N -> C |
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Term
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Definition
Occur between Cysteines
Must be broken before determining structure, by adding a reducing agent (DTT, Na2S2O4) |
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Term
Methods for Site-Specific Peptide Cleavage...
Trypsin
Chymotrypsin
Cyanogen Bromide |
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Definition
ENZYMATIC
Trypsin: after Lys or Arg
Chymotrypsin: after Phe, Trp, Tyr
CHEMICAL
Cyanogen Bromide: after Met |
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Term
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Definition
The study of the entire complement (all) of proteins found in a particular tissue/cell type.
The darker the spot, the more the growth on 2D gel electrophoresis followed by mass spectrometry. |
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Term
Homologs:
Paralogs
Orthologs
Signature Residues |
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Definition
Homologs: proteins with similar characteristics due to shared ancestry
Paralogs: found in the same organism/species
Orthologs: found in different species
Sig Res: can have gaps, but determine each individual identity; evolution basis |
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