Protein Purification: You have a tube containing trypsinogen, chymotrypsinogen and chymotrypsin in an aqueous buffer. Using atleast two methods of protein purification , propose a scheme for seperating the three proteins. Explain how each method works. physically or chemically. If you choose affinity chromatography, you must clearly explain your choice of affinity substrate.
Protein Purification: You have a tube containing trypsinogen, chymotrypsinogen and chymotrypsin in an aqueous buffer. Using atleast two methods of protein purification , propose a scheme for seperating the three proteins. Explain how each method works. physically or chemically. If you choose affinity chromatography, you must clearly explain your choice of affinity substrate.
For unlimited access to Homework Help, a Homework+ subscription is required.
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.