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Proteins can perform what 5 functions?
- Enzymes - catalysis
- Structural - Rigidity of the cells
- Motor proteins - Convert chemical energy to mechanical work
- Cell signaling and signal transduction - receptors, insulin, receptors, etc
- Transport - transport fatty molecules, etc albumin and hemoglobin
- Regulatory proteins - transcription factors for gene expression
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What is salting out of proteins?
- Salting out is based on the decreased solubility of proteins at high salt concentrations.
- Different proteins are precipitated at different salt concentrations.
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What does trypsin cleave after in polypeptides?
Arg (R) and Lys (K), unless followed by Pro (P)
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What does chymotrypsin cleave after in polypeptides?
Phe (F), Tyr (Y), Trp (W) unless followed by Pro
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What does elastase cleave after in polypeptides?
After Ala (A), Gly (G), Ser (S), or Val (V) unless followed by Pro (P)
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What does endopeptidase v8 cleave after in polypeptides?
Cleaves after Glu (E), even if there is a Pro (P) present!
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What is the pKa of Lysine (Lys, K)?
10.79
- Asp - D - 3.9
- COOH - 4.0
- Glu - E - 4.07
- His - H - 6.04
- NH3 - 8
- Lys - K - 10.79
- Arg - R - 12.48
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What is the pKa of Arginine (Arg, R)?
12.48
- Asp - D - 3.9
- COOH - 4.0
- Glu - E - 4.07
- His - H - 6.04
- NH3 - 8
- Lys - K - 10.79
- Arg - R - 12.48
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What is the pKa of Histidine (His, H)?
6.04
- Asp - D - 3.9
- COOH - 4.0
- Glu - E - 4.07
- His - H - 6.04
- NH3 - 8
- Lys - K - 10.79
- Arg - R - 12.48
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What is the pKa of Aspartic Acid (Asp,D)?
3.9
- Asp - D - 3.9
- COOH - 4.0
- Glu - E - 4.07
- His - H - 6.04
- NH3 - 8
- Lys - K - 10.79
- Arg - R - 12.48
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What is the pKa of Gluatamic Acid (Glu, E)?
4.07
- Asp - D - 3.9
- COOH - 4.0
- Glu - E - 4.07
- His - H - 6.04
- NH3 - 8
- Lys - K - 10.79
- Arg - R - 12.48
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What are proteins sensitive towards (including denaturing)?
- pH
- High temperatures
- Presence of proteases
- Oxygen (keep at high concentration)
- Freezing (glycerol - water freezes and structure may tear apart)
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What does the gel filtration method exploit for sequencing?
Based on size.
- Exploits difference in protein size
- Mixture of proteins run through a column packed with porous polymers
- -small proteins enter the polymer via pores and are retarded inside
- -large molecules travel through column without retardation. These are the first to be elucated.
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what is chromatography in general?
- Separation of proteins by taking advantage of:
- -size (gel filtration)
- -charge (ion exchange)
- -binding affinity to ligands ( affinity chromotography, ie Immobilized metal affinity chromotography)
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What does the Ion Exchange Chromatography exploit (anion)?
It exploits the charge differences in proteins.
- Anion exchanger consists of anions binding to cationic group in the column.
- The postive proteins were first to leave the column.
- Q-resin: R-NR'3+
- DEAE-resin: R-NHR'2+
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What does the Ion Exchange Chromatography exploit (cation)?
It exploits the charge differences in proteins.
- Anion exchanger consists of cations binding to anionic group in the column.
- The negative proteins were first to leave the column.
- S-resin: RCH2-SO3-
- CM-resin R-O-CH2-COO-
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What does the hydrophobic interaction chromatrography exploit?
It exploits hydrophobic interactions to purify nonpolar proteins
- Substrate used is lightly substituted with octyl or phenyl groups
- High to low salt concentrations used
Hydrophillic proteins are the first to leave
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What does affinity chromotagraphy exploit?
Specific binding is exploited.
- Exploits protein's ability to bind specifically but noncovalently
- -Specific ligands with high affinity
Proteins with no affinity to substrate pass first.
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What does immobilized metal affinity chromotagraphy (IMAC) exploit?
Exploids affinity of proteins for metal ions that are tagged with a polysistine (tag of 6 - 8 histines)
The His-tag has an affinity towards metal ions.
Untagged proteins are first to leave.
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What is electrophoresis?
Separation of molecules based on charge and size
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What does Polyacrylamide gel electrophoresis (PAGE) take advantage of?
Mass and charge.
- Mobility of small molecules is greater than large molecules (with same charge density).
- -High pH is used so nearly all proteins are net negative
More negative move towards positive electrode.
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What does SDS-PAGE exploit?
Separates by mass only.
- Disrupts tertiary structure by breaking noncovalent bonds
- -proteins unforld into rod-like shape.
Separation by mass based on distance traveled.
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What does capillary electrophoresis (CE) exploit?
High voltage to separate based on charge
- Fast and efficient separation with the use of high voltage
- Both positive and negative ions travel through capillary by electroosmotic flow.
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What does Isoelectric focusing (IEF) exploit?
pH gradient and electric field
- Protein's charge will change based on pH
- Electric field used to attract towards area but will stop when pH = pI
Two dimensional (2D) gel electrophorsis used IEF and SDS page to separate by mass and by charge (as a function of pH)
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What are immunoassays?
- Use of antibodies for specific binding and identification method.
- ie - coloring in western blot or ELISA.
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What are the steps for Enzyme linked immunosorbent assay (ELISA)?
- 1) Immobilize the first antibody on a solid support
- 2) Wash excess antibodies not adhered
- 3) Incubate with protein
- 4) Was excess protein
- 5) Expose to second antibody with assayable enzyme such as a detectable product.
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What is the principle of Western Blot (8)?
- 1) Separate proteins by polyacrylamide gel electrophoresis (PAGE)
- 2) Transfer proteins on nitrocellulose filter by electroblotting - the protein attach to the filter
- 3) Incubate the membrane with a protein-containing buffer (skim milk or casein) - fills open spots
- 4) Incubate the membrane with primary antibodies specific to the protein of interest. - specifically binds to the protein
- 5) Wash out excess of primary antibodies.
- 6) Incubate the secondary antibodies with conjugated with a label or enzyme. - affinity towards primary antibody (not protein like ELISA)
- 7) Wash out excess of secondary antibodies.
- 8) Detect the signal.
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