Protein Purification

AKTApurifier (GE Healthcare)AKTApurifier

The AKTApurifier is a large scale FPLC system capable of separating samples of 250 µl up to 2 ml using gel filtration techniques, or larger sample volumes (up to 50 ml) if utilising affinity chromatography. Fraction sizes range from 0.5ml up to 2ml. A variety of gel filtration columns to separate proteins and complexes of different sizes are available in the facility. This system is also suitable for the purification of tagged proteins (His, GST, MBP) via affinity chromatography, as well as ion exchange separations (cation and anion).

AKTAmicro

AKTAmicro / SMART system (GE Healthcare)

For analytic work, we have two Micro-FPLC systems capable of separating small sample volumes (up to 50 µl) when samples are precious or of limited availability. Fraction size is 50µl. These systems are mostly used for gel filtration separations for the analysis of proteins or complexes. The AKTA systems are housed in a refrigerated cabinet and the SMART system has its own refrigeration, so regardless of which system is used, samples can be separated at 5oC.

Method_2

 

Combination of protein purification methodologies with mass spectrometry

Protein purification techniques such as gel filtration can be effectively combined with mass spectrometry for analysis of protein complexes. Single fractions can either be analysed following SDS-PAGE and coomassie staining, or selected fractions can be combined, immunoprecipitated and the resulting sample separated on a coomassie gel or digested for MS analysis in solution. This approach provides very clean preparations for subsequent mass spectrometry.

In the example shown the cellular lysate has been fractionated using a Superose –6 gel filtration column.  The indicated fractions are pooled together and subjected to immunoprecipitation with an appropriate antibody. The proteins are eluted and separated using SDS-PAGE before analysis by LC-MS/MS.  This approach allows the proteomic composition of the complex to be characterised and can provide important knowledge as to how the particular complex is functioning.

This approach is ideal for analysing the formation of new complexes; a very good example is the apoptosome complex which can be assembled ‘in vitro’ and gel filtration experiments can identify the movement of the constituent molecules (Apaf-1, caspse-9 and cytochrome c) into the large apoptosome complex.

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