Lecture 2, microscopy - Methodology and microscopy !...

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Methodology and microscopy
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Figure 8-19 Molecular Biology of the Cell (© Garland Science 2008) SDS-PAGE is commonly used to separate and characterize proteins. SDS = s odium d odecyl s ulfate (a detergent) PAGE = p oly-a crylamide g el e lectrophoresis The example on the left shows the purification of a protein from an extract (lane1) to the final purified product (lane 5). Apparent molecular weight of purified protein is ~40,000 daltons.
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Figure 8-17 Molecular Biology of the Cell (© Garland Science 2008) (negatively) charged detergent reducing agent Detergents work because they are amphiphilic (or amphipathic) – partly hydrophilic (polar) and partly hydrophobic (nonpolar)
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Figure 8-18b Molecular Biology of the Cell (© Garland Science 2008) Functions as molecular sieve, with smaller proteins migrating faster. Protein can be detected by subsequent staining (e.g., with Coomassie blue) 2D gels also useful (with separation by pH in 2nd dimension) (mercaptoethanol is a reducing agent) The amount of SDS bound is very roughly proportional to length of protein (What if mercaptoethanol is not used?)
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Figure 8-18a Molecular Biology of the Cell (© Garland Science 2008)
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Figure 9-18 Molecular Biology of the Cell (© Garland Science 2008) Detecting a specific protein in a mixture of proteins (by immunoblotting or western blotting) Marker allows visualization of 2 nd antibodies (and hence antigen A). Often marker is a molecular that emits light.
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Lecture 2, microscopy - Methodology and microscopy !...

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