Role of Ethanol:ether

During the purification of Lapsi leaf protease we used the following steps:
1)20 gram of dried and finely grinded lapsi leaf powder was washed with acetone (75ml)twice
2)After this 5 gm of acetone washed powder was suspended in phosphate buffer(0.1m,ph7)for two hours.
3)After this the filtrate was collected.Volume was measured and was heated for half an hour for 30 minutes.
4)Then we added TCA of 2.45M to make the final concn.0.2M.
5)Then it was centrifuged at 10000 rpm for 10 min.
6)The pellet thus obtained was dissolved in 1 ml of phosphate buffer(0.1M,Ph 7).
7)Again 2.45M TCA was added to make the final concn.0.2M.
8)Then it was centrifuged for 10 min at 10000rpm.
9)The pellet thus obtained was dissolved in 0.5ml PB and then 0.5ml of 1:1 ethanol ether was added to it and was suspended for 1 hour.
10)Then it was centrifuged again at 10000rpm for 10 mins.
11)The pellet thus obtained was dissolved in 100ul of (1:1) ethanol:ether and was left overnight in the freeze.
12)Again it was centrifuged at 10000rpm for 10 mins.
13)The pellet thus obtained was dissolved in minimal volume of PB.



This is the protocal we have been following in the lab......but some questions still stikes on to my mind:

in step 3 above why do we have to heat the solution before adding TCA....?is it just to activate the protease?or can there be another reasons?
other question that strikes to my mind is why do we add ethanol :ether....is to remove the lipo proteins,polyphenols........?as far as i think addition of organic solvent to the protein denatures the enzyme......
But the most surprising fact we came to encounter is the enzyme is still active after all these steps..it still chops BSA at a vigorous rate.......

Lapsi

Doing the work on lapsi leaf protease........i am realizing how difficult the research
stuff is.Lapsi leaf protease as shown by earlier research and also with our experience is an acid loving protease..readily precipitates with TCA and it is unaffected by heat........a unique type of protease.
Research is getting more intresting the protese doesn't moves in gel in cationic native PAGE......and we too tried with anionic PAGE.....but yet haven't got the clear result or a band........how can we find a clue to it.......?still working on it hope we are close to finding something new and intresting.....for now me and Bikash are not having a clue......
Does TCA precipates things other than protein??the pellet that we obtained after precipitation is surely a protein

I have started my research work on purification and characterization of Choerospondias axillaris(lapsi )leaf protease.........