BIOCHEMISTRY:
28. If you place a protein with an oblong shape on a gel filtration column, why does it appear to be larger than a roughly spherical protein in its elution profile, even though the molecular weight of both proteins is the same? ______________________________
29. [T/F] A Sephadex G-25 gel filtration column can be used to separate alcohol dehydrogenase from β-amylase.
30. Explain how to determine the exclusion limit of a gel of unknown origin.
BIOCHEMISTRY:
28. If you place a protein with an oblong shape on a gel filtration column, why does it appear to be larger than a roughly spherical protein in its elution profile, even though the molecular weight of both proteins is the same? ______________________________
29. [T/F] A Sephadex G-25 gel filtration column can be used to separate alcohol dehydrogenase from β-amylase.
30. Explain how to determine the exclusion limit of a gel of unknown origin.
Related textbook solutions
Related questions
You are in the final step of a protein preparation in the biochemistry lab. At this point, you are left with a mixture of just four well-defined species in solution. The four protein species, as well as some of their chemical properties, are listed in the table below. Design a complete purification scheme to separate all the components of the mixture, and depict your separation scheme using a procedural flow chart.
| Species | Molecular weight | pI | Affinity tagged? |
| A | 27 kDa | 5.1 | 6xHis |
| B | 5 kDa | 5.0 | 6xHis |
| C | 33 kDa | 7.5 | 6xHis |
| D | 28 kDa | 4.8 | none |
Experimental information:
Size exclusion/gel filtration only works well as a separation method if the two proteins differ in size by more than a factor of 2.
The 6xHis tag is a series of six histidine residues that are engineered into the primary sequence of the protein. Proteins containing a 6xHis tag can be purified by nickel-NTA (Ni-NTA) affinity chromatography. This method is very common in modern biochemistry.
For ion exchange columns, clearly state the pH(s) for the column and whether you would use a cation or anion exchange column.
