Lecture 1, methodology and microscopy2

For technical reasons not all tryptic peptides are

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tryptic peptides with m/z values of e, g and i. (For technical reasons, not all tryptic peptides are detectable by mass spectrometry). Which protein do you think you have purified? MATCHES PROTEIN #2! You know you purified protein 2 and other a, b, c, etc., are unique numerical values
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Immobilization of a protein mixture (that has been separated by SDS-PAGE) on a surface (often a sheet of nitrocellulose membrane). A specific protein in a mixture of proteins can be detected and quantified by immunoblotting (a.k.a. western blotting) Why are enzyme-linked secondary antibody (Ab2) used and not just enzyme-linked primary antibody (A primary antibody, so more shows up when linked to secondary AMPLIFICATION Ab2 is typically linked to an enzyme that can emit light, which can be captured and quantified digitally.
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See Figure 8-14 Molecular Biology of the Cell (© Garland Science 2015) After SDS-PAGE, protein mixture can be transferred to a membrane. Specific proteins (e.g., X) can be detected by immunoblotting with antibody (e.g., one that is directed against protein X, as shown on the right) protein X (detected by light emitted by Ab2- linked enzyme; see last slide)
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If antibodies (anti-A and anti-B) directed against two different proteins (A and B) are available, how can we check whether these two proteins associate with each other (directly or through additional proteins)? A B A B C Immunoprecipitation (or co-immunoprecipitation), followed by SDS-PAGE and immunoblotting.
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Figure 9-1 Molecular Biology of the Cell (© Garland Science 2015) Figure 9-2 Molecular Biology of the Cell (© Garland Science 2015) Resolving power (resolution) is an important factor in the choice of microscope to be used. Magnification is generally less of a concern. The Nobel Prize in Chemistry 2014 was awarded to Eric Betzig, Stefan Hell and William Moerner "for the development of super-resolved fluorescence
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Figure 9-3 Molecular Biology of the Cell Garland Science 2015) A (conventional) light microscope (or CCD camera) adjustable for focusing Figure 9-36 Molecular Biology of the Cell (© Garland Science 2015) 2 point sources of light (from specimen) <200 nm apart will not be resolvable and will not appear as separate objects
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Figure 9-6 Molecular Biology of the Cell (© Garland Science 2015) Note that resolution here does NOT refer to the resolution of the image capturing device (typically a CCD camera). The better or “higher” the resolution, the lower the value of R (R) With violet light: ~ 400 nm < 90 sin < 1 R ~ 200 nm (or 0.2 m)
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When light passes through a cell, the phase of the light wave is changed according to the cell’s refractive index. The degree of phase shifting varies according to the refractive index and thickness of the part of the cell that the light passes through.
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  • Spring '09
  • BUSHART
  • molecular biology, Garland Science, Molecular Biology of the Cell

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