Specific activity during multi-step purification

[irrumator11]

Hi all, long-time reader, first-time caller.

 

I'm doing a multi-step purification to purify a protein (or proteins!) responsible for a particular biochemical activity. I do not know the identity of the protein, so I am doing the purification by following the activity only. Here is my purification table.

 

 

_____Step_____________________Total protein_____Specific activity ____Yield____Fold_________

__________________________________(mg)____________(units/mg)__________(%)___purification___

 

Processed crude fraction___________57.6______________35_______________100_______=1_____

Ammonium sulfate___________________36.9______________74_______________87________2.13____

Phenyl sepharose___________________6.64_____________1560______________29________44.9____

Hydroxyapatite______________________2.8_____________1463_____________7.9________42.1____

Heparin sepharose_________________0.46_____________2215_____________2.5________63.8____

Gel filtration____________________0.082_____________2356_____________1.9 _______67.8____

 

 

As you can see, something funky is happening with the Hydroxyapatite column, because the specific activity does not increase at that step. I am stumped as to what that can mean. Is the hydroxyapatite column fractionating out a cofactor or another protein that is necessary for activity? This is my first time doing a purification like this. Is a yield of 1.9% considered colossally shitty? It seems I am losing the most yield at the Phenyl sepharose step -- does that mean I should eliminate that column from my purification?Any help is appreciated! Thanks in advance!