Lecture9 Notes

Lecture9 Notes - Lecture 9 Solution Characterization of...

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Unformatted text preview: Lecture 9 Solution Characterization of Polymers III ANNOUNCEMENT: Review Session will be Tuesday during normal lecture time. Equation sheet and practice exam are posted Exam will cover lectures 1-9 1 Wednesday, October 20, 2010 Technique Information Comments SEC HPLC Osmotic Pressure Viscosity MALDI, Mass Spec Fractionation Ultracentrifugation End Group Analysis Cryoscopy, Ebulliometry VPO SLS DLS 2 Wednesday, October 20, 2010 Dilute Solutions of Biomacromolecules Why are biomacromolecules difficult to work with? 3 Wednesday, October 20, 2010 Gel Electrophoresis •Gel electrophoresis is used to separate macromolecules by charge •The gel is crosslinked polymer - poly(acrylamide) for proteins and small nucleic acids, and agarose for larger nucleic acids (>100 bp) • • • • • Electromotive force is used to push or pull the charged molecules through the matrix Molecules with a negative charge move towards the anode, and a positive charge towards the cathode At the end of the run, the smallest molecules reach the anode/cathode, the gel is stained to make the macromolecules visible Typical stains include coomassie blue, ethidium bromide, silver, or the analytes may fluoresce The distance traveled is proportional to the molecular weight, and molecular weight markers are used to “calibrate” the gel 4 Wednesday, October 20, 2010 SDS-PAGE • • • • • • • SDS-PAGE: Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Used to separate proteins according to electrophoretic mobility, which is a function of length, molecular weight, higher order protein folding, posttranslational modifications etc SDS denatures secondary and non-disulfide linked tertiary structures, applies a negative charge to each protein in proportion to its mass Without SDS, different proteins with similar MWs would migrate differently due to differences in folding (hydrodynamic size) Stacking gel has large pores, concentrates proteins into a small band Resolving gel has small pores, separates based on size Proteins can also be reduced to disrupt covalent tertiary structures 5 Wednesday, October 20, 2010 Immunoblot • • • • • • Method used to detect a specific polymer of interest Gel electrophoresis is used to separate the polymer of interest, which then may be transferred to a membrane where antibodies are used to specifically detect it Western Blot used to detect proteins (far Western uses non-antibody protein; detects protein-protein interactions) Southern Blot used to detect DNA; uses agarose gel electrophoresis to separate DNA, which is then transferred to a filter membrane for probe hybridization (DNA is cut into smaller pieces, which are separated on the gel and then transferred to a membrane. DNA is detected with hybridization probes, which are labeled) Northern Blot is used to detect RNA in the same method as DNA, in order to study gene expression Southwestern blot is used to characterize proteins that bind to DNA- the proteins are separated and transferred to a membrane and stained with labeled DNA 6 Wednesday, October 20, 2010 ELISA • • • • • Enzyme linked immunosorbent assay Protein or polymer of interest is immobilized on a surface (specific or non-specific) Detection AB is added; primary AB is covalently linked to an enzyme, or secondary AB is linked to enzyme An enzymatic substrate is added to produce a visible signal which indicates the quantity of the antigen Substrate can be chromogenic, or more commonly now, fluorogenic A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form. 7 Wednesday, October 20, 2010 Vroman Effect 8 Wednesday, October 20, 2010 Circular Dichroism • • • If a molecule is chiral or asymmetric, it will absorb left-and right-circularly polarized light differently CD (absorptive) is observed for λ where the compound absorbs light; at other λ, optical rotation (dispersive) occurs Optical rotation is a consequence of a different RI for left- and rightcircularly polarized light 9 Wednesday, October 20, 2010 NMR •Detects nuclei with magnetic moments (1H and 13C most common) •Nuclei flip in a magnetic field of the correct strength •Position in ppm (0 for TMS internal standard) •used to obtain information about the structure and dynamics of proteins 10 Wednesday, October 20, 2010 Proteins • • • • • • • Proteins can be separated via selective precipitation using salt solutions The solubility of proteins in aq. buffer depends on the distribution of hydrophilic and hydrophobic AAs Repulsive forces prevent aggregation and promote dissolution; ions aggregate around the hydrophilic areas, and water around the hydrophobic areas Changing the salt concentration destabilizes the protein in solution and compresses the solvation layer, increasing protein-protein interaction As salt is increased, less (non-ionized) water is around the solvate the protein and the hydrophobic patches start to aggregate This results in a small decrease in the enthalpy and a larger decrease in entropy When the water molecules forming the solvation layer around the protein are brought back into the aq phase, the entropy increases and ΔG becomes negative and the protein spontaneously precipitates analogous to fractionation 11 Wednesday, October 20, 2010 Exercise Working in groups, discuss which method you would use to gain the following information: 1) Mn, Mw and pdi of a poly(caprolactone) scaffold as it degrades into smaller fragments; the degradation is correlated with the release of a drug 2) The size of cross-linked poly(styrene) beads you will use in your microfluidic device 3) The molecular weight of a protein growth factor you expressed in the lab 4) The conformation of a synthetic polymer in three different solvents, to determine excluded volume 5) Separation of different proteins expressed by a cell in response to a stimulus 6) The molecular weight and purity of short polypeptides used for integrin binding experiments 7) The efficiency and of conjugating diptheria toxin (Mn ~60,000) to a transferrin (Mn ~ 70,000), and separation of conjugated and non-conjugated products 8) The Mn and Mw for polyethylene with long branches 12 Wednesday, October 20, 2010 Practice Exam 13 Wednesday, October 20, 2010 ...
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