To make full profit of these activities as learning tools, you are advised to work them out using the simulator, rather than solving them as written theoretical problems or just looking at the answer straight away.
As these are problem-solving activities, not textbook-style questions, you should seek out of this website any information you may need about the properties of the chromatographic matrices or the proteins. However, experimentation will be the best way to learn.
Using several gel filtration matrices, determine the relative order of elution of these proteins: aldolase, aprotinin, conalbumin, ferritin, glucose-6P-dehydrogenase.
| drag the names within the list to reorder them | elutes first
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When done,
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a) Which is the electric charge of ovalbumin and aldolase in borate buffer?
| select your chosen option | negatively charged
uncharged
positively charged
|
When done,
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b) Which ion exchange resin could you use so that both are retained (>15 mℓ) in the column, being resolved from ribonuclease A?
| select your chosen option | CM-cellulose
DEAE-cellulose
|
When done,
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Observe the separation of a mixture of thyroglobulin, glutathione reductase and carbonic anhydrase, using Sephacryl 100 and 300 (size exclusion / gel filtration matrices), and deduce: Which one has a larger molecular size? Is that molecular mass above or below 200 kDa?
| select your chosen option The largest protein is... |
thyroglobulin
glutathione reductase
carbonic anhydrase
|
When done,
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| select your chosen option Molecular mass is... |
>200 kDa
<200 kDa
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When done,
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Find a chromatographic matrix to achieve good separation of the 3 components in a mixture of thyroglobulin, ribonuclease A and ferritin.
| select your chosen option | When done,
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a) Which protein(s) are retained in a column filled with IR-Sepharose 4B?
| drag to the left column those that are retained,
to the right those that are not |
retained:
|
not retained:
|
When done,
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b) Which ion exchange resin could be used to retain it/them at pH=6.8?
| select your chosen option |
DEAE-cellulose
CM-cellulose
|
When done,
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a) Which proteins are retained in a column filled with Con A-Sepharose 4B? Is any of them retained more strongly than the others?
| drag to the left column those that are retained (and put them in order),
to the right those that are not |
retained:
most retained
|
not retained:
|
When done,
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b) What is the reason?
(open-ended response)
Which feature is shared by proteins that are retained on 2’5’ADP-Sepharose 4B?
(open-ended response)
Which protein(s) are retained on Ni2+-HiTrap Chelating HP?
| drag to the left column those that are retained,
to the right those that are not |
retained:
|
not retained:
|
When done,
|
Given a mixture of three proteins, A, B and C, with isoelectric points pIA = 4.2, pIB = 7.0 and pIC = 7.7, could you separate them using a single chromatographic run?
(open-ended response)
Purification of a protein usually requires more than one chromatographic step. Let's consider you are working in a lab and need to purify DNA polymerase from a bacterial culture. Since you know the molecular mass of this protein, you have performed a gel filtration chromatography of the cell lysate. After that you want to run an ion exchange step. Would you select DEAE-cellulose or CM-celullose as the resin for chromatography? Justify your answer. Later on, you decide to enhance purification doing affinity chromatography. Which immobilised ligand would you use in order to retain the protein of interest?
(open-ended response)