1. Assume a mixture of GFP, cytochrome c and hemoglobin was separated using the
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1. Assume a mixture of GFP, cytochrome c and hemoglobin was separated using the same gel filtration column and conditions you used in lab. Based on the size of each protein, list them in order from smallest to largest expected partition coefficient value. 2. Justify your answer above. 3. Are any of the proteins larger than the exclusion limit? If so which ones? Concisely explain the expected value for partition coefficient for a protein larger than the exclusion limit. 1. Assume a mixture of GFP, cytochrome c and hemoglobin was separated using the same gel filtration column and conditions you used in lab. Based on the size of each protein, list them in order from smallest to largest expected partition coefficient value. 2. Justify your answer above. 3. Are any of the proteins larger than the exclusion limit? If so which ones? Concisely explain the expected value for partition coefficient for a protein larger than the exclusion limit. 2. Justify your answer above. 3. Are any of the proteins larger than the exclusion limit? If so which ones? Concisely explain the expected value for partition coefficient for a protein larger than the exclusion limit.Explanation / Answer
In the above given mixture, GFP is green flourescent protein which has molecular weight of 27KDa or 27000 daltons. the molecular weight of haemoglobin is 64,458 daltons. the molecular weight of cytochrome c is 12000 daltons.
the large size molecules does not permeate into the stationary phase pores and hence will elute faster. whereas the small size molecules permeate into the pores of the stationary phase or solvent and elute slower. hence , small size molecules will have greater partition coefficient.
in the above mixture, the order from smallest to largest according to expected partition coefficient will be: haemoglobin, GFP, and cytochrome c.
the partition coefficient for small size molecule will be explained by: Ve - Vo / Vi
where Ve - Vo is extra stationary phase that molecule can accesss.
Vi is extra pore volume.
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