Jump to content

Specific activity during multi-step purification

Featured Replies

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!

Archived

This topic is now archived and is closed to further replies.

Important Information

We have placed cookies on your device to help make this website better. You can adjust your cookie settings, otherwise we'll assume you're okay to continue.

Configure browser push notifications

Chrome (Android)
  1. Tap the lock icon next to the address bar.
  2. Tap Permissions → Notifications.
  3. Adjust your preference.
Chrome (Desktop)
  1. Click the padlock icon in the address bar.
  2. Select Site settings.
  3. Find Notifications and adjust your preference.