Question

Biochemistry: Chromatography

You are trying to separate protein A from four other proteins (B, C, D, E). These proteins have the following properties. (MW is in daltons and charge is at pH 7):

Protein A (GST-tag, MW: 18,000, charge +24)

Protein B (MW: 64,000, charge -10)

Protein C (His-tag, MW: 120,000, charge +6)

Protein D (MW: 84,000, charge 0)

Protein E (MW: 7,000, charge -16)

Below, name three separate techniques you can use to attempt to separate protein A. For each technique, explain what property of protein A the technique is using/ exploiting. Also, for each technique, list the order in which the proteins will elute (from first to last).

Answer 1

THE 3 SEPARATE TECHNIQUES TO SEPARATE PROTEIN-A :

1. ION EXCHANGE CHROMATOGRAPHY
2. GEL-FILTRATION OR SIZE EXCLUSION CHROMATOGRAPHY
3. AFFINITY CHROMATOGRAPHY

Properties of PROTEIN A that the 3 above techniques are using and their order of elution:

1. ION EXCHANGE - THE NET CHARGE ON THE PROTEIN IS CONSIDERED. ( Here the column is either cationic or anionic. For instance: If the column is cationic ( Positively charged), the elution rate of the above proteins would be PROTEIN-A, PROTEIN-C, PROTEIN-D , PROTEIN-B & PROTEIN-E. This is because, the protein with the same charge would be eluted out first, and the ones with the opposite charge would be eluted out last. Remember Proteins with Opposite charges attract to the column and those with the same charge as the column would be eluted out first.)
2. GEL FILTRATION/ SIZE EXCLUSION - THE Size- MOLECULAR WEIGHT ON THE PROTEIN IS CONSIDERED. ( Looking at the molecular weight (MW), one can guess the bulkiness of the protein. Thus, the elution rate would be the less bulky proteins come out first and the most bulky proteins come out last. ) Order from 1st to last would be: PROTEIN C, D, B, A & E .
3. AFFINITY CHROMATOGRAPHY - THE AFFINITY OF THE LIGAND THAT IS ATTCHED ON THE PROTEIN IS CONSIDERED. ( In the above case, since we look to separate PROTEIN A, notice that it has a tag of GST. Thus we need a column that is capable of retaining GST and elute out the remaining 4 proteins.