1. Assume a mixture of GFP, cytochrome c and hemoglobin was separated using the
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Question
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. (the exlcusion limit is 80,000).
Explanation / Answer
Q.1 Answer- Since the size of GFP, cytochrome c and hemoglobin are 27 kDa(kilo Dalton) , 12 kDa and 130 kDa respectively. So the order to list them from smallest to largest would be Cytochrome c < GFP < Haemoglobin.
Q.2 Answer- Gel filtration chromatography is a method in which molecules in solution are separated by their size & in some cases by molecular weight. Separation is achieved using a porous matrix to which the molecules have different degrees of access. Thus small molecules have greater access and larger molecules are excluded from the matrix. As per this degree of access , cytochrome c will move through more faster than GFA ,while Haemoglobin molecules would have least access through it.
Q.3 Answer- Since the given exclusion limit is 80000 or 80 kDa therefore None of the above given proteins is larger than this exclusion limit.
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