Question

Purification & Size Determination of GFP & BFP ment estions 1. What is the anticipated difference in apparent molecular weight between pFluoro- Green" (gfp) and pFluoroBlue™ (bfp) as detected by denatured SDS- polyacrylamide gel analysis? 2. Why is the molecular sieving matrix swelled prior to packing the column? 3. What is the basis of molecular sieve chromatography? 4. Can molecular sieve chromatography columns be used to separate DNA fragments?

Answer 1

1.The difference between the two proteins is the substitution of two amino acids. This difference in mass is negligible and therefore the two proteins will be identical in molecular weight as judged by this method of analysis.
2.  When the matrix is swelled in an aqueous buffer it will form spheres which provide the sieving property for the separation of biomolecules.
3.Molecular sieve matrices are based on the separation of biomolecules based on size and shape. Assuming such molecules are globular, then size is the major parameter for
the separation. If the biomolecules are too large to penetrate the pores in the molecular sieve, the large molecules will go around the beads and will elute in the exclusion
buffer with no separation. By contrast molecules that penetrate the Sephadex beads will be fractionated based on size with large to smallmolecules eluting sequentially.
4. Small fragments of DNA (oligonucleotides) can be separated by molecular sieve chromatography. DNA molecules are large for fractionation using most Sephadex
matrices and therefore DNA will not penetrate the beads. For example a 1 Kb double stranded DNA fragment is 2000 nucleotides when multiplied with the molecular weight of 500 for the average of a nucleotide is 1,000,000 daltons. Unlike DNA, protein molecules tend to be much smaller for example, the gfp and bfp are bout 40,000 daltons and are therefore ideal candidates to be separated by a molecular sieve type of chromatography.