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.
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.
