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BIO 201: Exam 2
Electrophoresis
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A technique for separating molecules (such as DNA fragments) from one another on the basis of their electric charges and molecular weights by applying an electric field to a gel.
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Anode
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Positive pole
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Cathode
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Negative pole
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What does an anode attract?
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Anions (negative charges)
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What does a cathode attract?
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Cations (positive charges)
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What can electrophoresis be used for?
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To separate, purify and identify proteins, RNA or DNA
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Native gels
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No detergent added
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What affects the migration of native gels?
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Native Conformation (shape), size, solubility and charge
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Detergent (usually SDS) gels
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Detergent added to the sample and the gel
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What affects the migration of detergent gels?
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Size ONLY
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What does SDS-PAGE stand for?
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Sodium dodecyl sulfate- polyacrylamide gel electrophoresis
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What part is the gel/ what part is the detergent?
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Polyacrylamide is the gel and SDS is the detergent
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What happens in SDS-PAGE?
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All polymeric protein complexes will be broken down into their monomeric forms
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Is SDS an anionic or cationic detergent at most pHs used?
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Anionic
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SDS separates proteins on the basis of:
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Size
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What size proteins move through the gel faster?
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Small
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What should you assume about the proteins in the presence of SDS?
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Denatured, negatively charged and soluble
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With no detergent present, what is the charge and shape of the proteins?
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Proteins could have a net charge of positive, negative or zero and be in their native conformation, depending upon the pH
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Weisenburg method: why are there other proteins besides tubulin present & how would you test this?
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They might reattach by ionic bonds after repolymerization, add high salt during the weisenberg procedure and you will only see tubulin
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Where are axonemal microtubules found?
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Eukaryotic cilia
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What is the pattern of microtubules?
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"9+2" = 9 pairs of fused microtubules and 2 infused inner microtubules
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How many tubulin chains does eachg microtubule consist of?
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13
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Motor protein for microtubules:
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Dynein
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Linker protein for microtubules:
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Nexin
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Pericentriolar material
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Material around the centrioles
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What is the function of the pericentriolar material?
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Serves as a Microtubule Organization Center (MTOC)
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What makes a centromere?
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Two centrioles
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Fluorescent protein labeling technique
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The fluorescent compounds are part of the proteins to make them fluorescent
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What does the fluorescent protein technique label?
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Express in cells, only newly made polymers
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Fluorescent antibody labeling technique
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Assume that one type of antibody only binds to one type of protein. Make that antibody fluorescent, add it to the cell and it will show you where the protein is in the cell
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What does fluorescent antibody technique label?
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ALL polymers (both old and new) that contain that protein with fluorescent antibodies
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