BIOL 200 Lecture Notes - Lecture 28: Mass Ratio, Electrophoresis, Differential Centrifugation
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Differential centrifugation separates cell components according to
Mass | |
Sedimentation rate |
Density | |
Charge |
Density gradient centrifugation separates cell components according to
Density | |
Sedimentation rate |
Mass | |
Charge |
If you are presented with a graph with many fractions on the x-axis, or a western blot where the lanes were from many fractions, what method was used?
Affinity purification | |
Western blot |
Density gradient centrifugation | |
Differential centrifugation |
What is the principle behind denaturing protein gel electrophoresis?
Proteins are separated according to type of amino acid R groups | |
Proteins are separated according to charge |
Proteins are separated according to size |
In denaturing gel electrophoresis, proteins that run farther down the gel are:
Smaller than those at the top of the gel | |
Larger than those at the top of the gel |
What is a western/immunoblot?
All proteins are stained with a general protein stain | |
Specific proteins are identified by antibodies |
Specific proteins are purified and detected by a general protein stain |
What is the principle behind affinity purification?
Specific molecules are removed from solution by a high affinity interaction on a solid support | |
Specific molecules have an exclusive sedimentation rate that be exploited with differential centrifugation |
Specific molecules have an exclusive size that can be exploited with SDS-PAGE |
Which of the following is quantification of variance in an experiment?
Median | |
Error bars |
Mean |
You have been given a blood sample from a patient with a bleeding disorder. You need to purify thrombin (a blood clotting enzyme present in blood serum) so that it can be characterized.
1) The first step in the purification is to separate serum proteins, including thrombin, from the whole cells present in the blood sample. What method could you use for this separation? What fraction of the resulting separation would you save?
2) You have managed to separate thrombin from all but two other serum proteins (see the table below). You run the proteins through an anion exchange column using a buffer at pH 6.4.
Protein | pI | Size (kDa) |
Cytochrome c | 10.6 | 12.0 |
Lactoglobin | 5.2 | 18.4 |
Thrombin | 7.1 | 36.0 |
i) Which protein(s) remain(s) bound to the column?
ii) How could you elute thrombin and achieve proper separation?
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