Lectures for Electronic Spectroscopy including CD

Lectures for Electronic Spectroscopy including CD - END...

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END Vibrational Spectroscopy START Electronic Spectroscopy
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Electronic (Optical) Spectroscopy 0 012 34 ... ½h D e D x excited electronic state ground electronic state note that the excited state potential well is shifted to a different equilibrium bond length – this may be less or more than the ground state here it is also more shallow with respect to E For a transition such as this, we assume that the nuclei stay fixed while the electrons move This is the Born-Oppenheimer approximation
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0 012 34 ... ½h D e D x excited electronic state ground electronic state in these drawings, the x-axis is typically not just a bond length between two atoms It is, instead, a normal (vibrational mode). Note that the vertical line drawn (the excitation from the lower state to the upper state), is from the lower v=0 state to the v=4 (or so) state in the upper level. We look to where there is maximum spatial overlap between the lower and upper state wavefunctions. This is called the Franck-Condon principle
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Electronic Spectroscopy in Biochemistry has many variants Absorbance this is a common way to measure the concentration of proteins in solution Protein concentrations are typically measured via their absorbance at 280 nm one is measuring the numbers of these amino acids at 280 nm; the assumption is that every protein has a statistical number of these amino acids absorbances of some proteins Bovine serum albumin (BSA): 63 Bovine, human, or rabbit IgG: 138 Chicken ovalbumin: 70
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Absorbance in Protein Detection Assays Horse Radish Peroxidase (HRP) HRP is a 44kD glycoprotein It has 4 lysine residues that are used for bioconjugation (we get to this shortly) HRP oxidizes this substrate probably producing NO 2 groups at the amine sites results in a blue color
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Engvall E, Perlman P (1971). "Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin G". Immunochemistry 8 (9): 871–4. doi : 10.1016/0019-2791(71)90454-X Standard Absorbance (or fluorescence) ELISA Assay This is the ONLY protein assay that is reproducible enough to yield quantitative values for clinical diagnostics
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Optical Methods Labeling Approaches (cont'd) Optical Spectroscopies
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This HRP-linked antibody may have 10 or more biotin groups
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biotin 4 biotin binding sites Streptavidin is a tetramer with 4 binding sites for biotin binding affinity is better than 10 -13 M
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Affinity Measurements K D (in Mol units) describes the affinity of an antigen for an analyte An antigen might be a protein; an analyte an antibody K D = the concentation of antigen when 50% of the antigen is bound to the analyte The biotin/Streptavidin interaction (10 -13 M) is practically an irreversible event
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For a protein assay, typically the HRP-linked antibody is initally loaded up with biotin groups Introduction of Streptavidin-HRP conjugates results in many HRP conjugates being directly associated with the detection antibody - HRP is itself an enzyme, and can turn-over many copies of its own substrate molecule – so we get two stages of signal amplification.
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N-hydroxysuccinimide (NHS) =O an NHS ester of biotin H B R'-NH 2
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Lectures for Electronic Spectroscopy including CD - END...

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