Protein purification by two stage chromatography - MDH by anion exhange & gel filtration (Nov/19/2008 )
Biochemistry - First Year and already its pretty nails.
We have been given a calculation question to do for a tutorial on purification of malate dehydrogenase using anion exchange and gel filtration chromatography.
We are given a table. It contains data for the initial crude extract, giving values for volume (ml), MDH activity (Vmax)(micromol/min/ml) and Protein (mg/ml). We are then given the same values as recorded after each stage, so a set for anion exchange and a set for gel filtration.
We have been told to calculate the specific activities of MDH in the initial extract and after each purification step. I did this by dividing MDH activity by Protein in each row.
Secondly, to calculate percentage recoveries, which i used total protein in eluate/ initial protein x 100 for each step.
Thirdly, we had to work out degree of purification after each step which i used specific activity of eluate/ initial specific activity.
Now we have to work out kcat and concentration of MDH subunits in the final sample, if each subunit is assumed to weight 30 kilodaltons. I'm not sure about my previous calculations, but for this one I don't enough no where to make a start/ guess, so any help would be greatly appreciated!
percent recovery would be (total activity in eluate/initial activity)x100