HOME WORK ASSIGNMENT 2 – KEY
1. The protein mixture of X, Y and Z is first run a gel filtration column.
This column is used to separate the protein X from the lower molecular weight proteins Y and Z as flow through. The elutions from this column containing protein y and z can be separated with the affinity column chromatography or ion exchange chromatography.
2. The quality of the protein can be checked on a SDS-PAGE for contamination of other proteins or degradation of the same protein. Western analysis can be done to check degradation of the protein but contamination cannot be detected as antibody will be specific to the protein.
The quantity of the protein can be measured using Bradford assay or by running a SDS-PAGE with known concentrations of BSA along with your protein and then quantifying them using excel or any other software.
3. The protein Z must have been present in three forms, monomer, dimer and trimer. This proteins has tendency to be in dimeric or trimneric form therefore the intensity of the monomer band is so less. When the proteins are loaded after heat denaturation, dimeric and trimeric forms would be denatured and will form monomers resulting in single band.
4. A gel filtration chromatography under native conditions can be used to separate different forms of protein. Also density gradient centrifugation can be used.
5. The frictional co-efficient of fibrinogen is about 2.5. Fibrinogen has an elongated structure, the protein molecule will not enter the pores and will be eluted out faster than expected for a protein of that molecular weight. Therefore molecular weight determination with this method is skewed. So the correct size for this protein can be determined using SDS-polyacrylamide gel electrophoresis as the protein is completely denatured and will migrate as per its actual molecular weight.