MODIFIED TAP PROCEDURE AS OF 2/24/03


2 g (wet weight) of Renografin gradient isolated kinetoplast fraction in 8 ml of lysis buffer.
Add 0.4 ml 10 % NP40 ( to give 0.5 % final concentration).
Ice for 15'.
Sonicate 5" 3x , with 2' intervals on ice.
Centrifuge at 90K  for 10'.
Remove supernate.
Resuspend pellet in 5 ml of lysis buffer without detergent and re-extract the pellet.
Centrifuge again.
Pool both supernates ~ 12 ml.
Add supernate to 0.8 ml of washed IgG sepharose.
Keep at 4 o C with rotation for 17-19 hours.
Transfer to 2 x 1 ml columns
Reload solution.
Wash with IgG wash buffer with 3 columns volume.
Wash with TEV cleavage buffer with 2 x column volume.
Close bottom of the column and add 2.5 ml of TEV  cleavage buffer.
Add 250 units of TEV.
Seal column and incubate ON at 4 o C with rotation.
Open column and collect the flowthru.
Add stock CaCl2 to 2 mM final concentration.


Wash Calmodulin beads with Calmodulin binding buffer ( ~ 0.8 ml of beads).
Add the TEV released solution to the Calmodulin beads.
Incubate at 4 oC with rotation for 3-4 hours.
Transfer to 1 ml column and reload 3 x.
Wash with binding buffer with 4 columns volume.
Add 200 ul of Calmodulin elution buffer.
Let beads go into the column with the bottom closed off.
Add  5 ml of elution buffer.
Incubate for 30' with mixing.
Collect the flowthru by centrifugation.
Concentrate if necessary. Have used both Pierce micro dialysis cassettes and Millipore centrifugal
concentrators. 

Most of the loss occurs in the Calmodulin release step.

The TAP-tagged fusion protein can be detected using the PAP reagent since Protein A binds to IgG.
The PAP reagent is from Sigma - Cat no. P2026. We first block the membrane with 5% non-fat milk in PBS.
Then we treat with 1:2000 of the PAP reagent into PBS with 0.1% milk.
Then rinse well with PBS. Develop using the Pierce reagent SuperSignal West Pico
(Cat no. 34080). Expose to X Ray film.