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Amino Acids
Can you give the 1-letter and 3-letter names for all 20 amino acids within 5 minutes?
Can you draw a oligopeptide of any given sequence?
With correct stereochemistry?
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Amino Acids:Polar, Charged
Be able to draw,name and give1-letter/3-letter
codes for all 20
amino acids!
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pKa and pI
• pI = Isoelectric pointthe pH at which a molecule carries no net electric charge
pKa1pKa2
pI = [pKa1 + pKa2]
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1
2
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Torsion Angles Defined…
• Phi: angle made from atomsCcarbonyl,n-1-Nn-Calpha-Ccarbonyl,n
NH
NN
O O
OH
H
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Alpha Helix
Sequence coils in a right-handedmanner.
Notice hydrogen bonding along the helical axis from carbonyl oxygen
(of residue n) to the amino hydrogenof residue (n+4).
Hydrogen bonding stabilizes the alpha helical structure!
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Affinity Chromatography
Affinity What could be to cause proteinsto elute off of anaffinity column?
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Ways to Assess Protein Purification
• Total Protein Concentration Assays– Beer’s Law– Colorimetric Assays
• Specific Protein Assays– Activity Assays– Immunoassays
• ELISA• RIA
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Beer’s Law
€
A = εbcWhat is the molar concentration of a
solution of Bovine Serum Albumin (BSA) that exhibits
an A280 of 0.75 with a path length of 1 cm?
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Beer’s Law
€
A = εbcWhat is the molar concentration of a
solution of Bovine Serum Albumin (BSA) that exhibits
an A280 of 0.75 with a path length of 1 cm? Conc. =
0.75(43,824 M-1cm-1) x (1 cm)
Conc. = 0.00001711 M = 17 µM
Must know the value of
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ε
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Bradford Protein Assay
Suppose you have a protein mixture and you need to determine the protein concentration.
You cannot use Beer’s Law.Because you would not know the extinction coefficient
for the protein mixture at 280 nm
Alternative approach: Bradford Protein Assay
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Bradford Protein Assays
How is data generated?
How is data analyzed?
Bradford Protein Assay reagent
contains Coomassie brilliant blue which reacts with basic
(esp. Arg) and aromatic amino acids to yield a blue
color with intensity proportional
to the protein concentration.
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Data Generation: Bradford Assay
Reference contains only buffer
Create a series of protein standards at known
increasing concentrations and mix with the Bradford reagent
2.5 5 10 15 20 25 µg/mL
Treat the protein mixture of unknown concentration
in the same manner as the standards and compare -
visually and spectrophotometrically!