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19 Cards in this Set
- Front
- Back
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Purpose of beads
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Pourous nature
Small proteins can diffuse freely throughout the bead |
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What is the underlying assumption in determining the Mr from GEC
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That both the unknown proteins and the standards have the same molecular shape
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advantage of gel exclusion chromatography to gel electrophoresis
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under ideal conditions charge plays no role in the separation so that molecular weights of the native, undenatured proteins can be determined more easily
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Does GEC determine the subunit or native Mr?
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Native Mr
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What is the bead made of?
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Dextran-starch and cellulose
high Mr polymer of glucose |
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In GEC how is the velocity of the protein measured?
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volume eluted or time(as long as pressure is constant)
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What does the pore size of the bead depend on?
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polysaccharide concentration within the beads (concentration of sugars within the beads)
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When the proteins enter the beads, they are in what phase?
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Stationary
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What defines the speed in the mobile phase?
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pressure of the buffer being pumped by the FPLC (Fast Pressure Liquid Chromatography)
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How is the elution of the proteins from the column detected?
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UV abs at 280nm-Tyr and Trp
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What is the pressure defined as?
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volume of solution/time
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What's a condition to be a good standard?
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Proteins of known native Mr
Must have variable probability of entry into the beads |
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Why is a dye not needed to visualize the standards like the SDS-Page?
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The proteins themselves serve as visual markers since they have iron within their heme groups (Ferritin, Hemoglobin, Cytochrome C)
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What serves as the void volume control?
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Ferritin
Indicates start of elution of the initial sample. Not included in molecular weight determination |
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Reason for centrifugation and filtration
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to remove insoluble debris that could clog the column and to remove bacterial cells or dust
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How does air interfere with the column?
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air in the column could cause gaskets in the pump to break. Air in the column results in collapse of beads. Beads collapse, but may not result in large cracks, but resolution is reduced.
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How do you remove bubbles from the syringe
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invert syringe and tap out bubbles
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If you use time to determine protein velocity what must you convert it to?
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seconds
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What are the two elution profiles that you must run?
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elution profile with 5 standards
elution profile with 5 standards and unknown |
