Seraphin Lab TAP method


Séraphin Lab

TAP protocol




Seraphin www-Server From the Séraphin lab at the European Molecular Biology Laboratory (EMBL).

Content

Buffers



Beads preparation

Use 200mL of IgG agarose bead suspension (SIGMA A2909) or IgG sepharose (PHARMACIA 17-0969-01) for the first step and 200mL of calmodulin beads supension for the second step (STRATAGENE #214303).

Use a small column (e.g., Econocolumn, Bio-Rad) to wash the beads with 5 mL of IPP150 (IgG beads) or 5 mL of IPP150 calmodulin binding buffer (calmodulin beads).

Binding to IgG beads

To 10 mL of extract, corresponding approximately to 2L of yeast culture, add :

Rotate with IgG beads for 2 hours at 4oC in the column (close top and bottom of the column).

TEV protease cleavage

Remove the top plug first, then the bottom plug and allow the column to drain by gravity flow.

Close the bottom of the column and add 1mL TEV cleavage buffer and approximately 100 units of TEV enzyme (e.g., GIBCOBRL). Close the top of the column and rotate 2 hours at 16oC.

Remove the top and bottom plugs of the column and recover the eluate by gravity flow. The solution remaining in the column dead-volume may be eluted with an additional 200mL of TEV cleavage buffer.

Binding to, and elution from, Calmodulin beads

To the previous 1 mL eluate add 3 volume of calmodulin binding buffer (3 mL) and 3mL CaCl2 1M per mL of IgG eluate to titrate the EDTA coming from the TEV cleavage buffer.

Transfer this mix into the column containing the washed Calmodulin beads. After closing the column, rotate for 1 hour at 4oC. After binding allow the column to drain by gravity flow.

Wash with 30 mL IPP150 calmodulin binding buffer.

Elute 5 fractions of 200mL with IPP150 calmodulin elution buffer.

Protein analysis

To analyse proteins on a gel, precipitate the final eluates and some of the controls with TCA (Ozols, J. (1990) Methods in Enzymology 182, 587-601) and load on an exponential 7/20% SDS-PAGE gel.

For complementation and functional studies dialyse against an appropriate buffer and keep at -80 oC.



Reference

Publication reporting results obtained with the TAP strategy should refer to:

A generic protein purification method for protein complex characterization and proteome exploration
Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M. and Séraphin, B.
Nature Biotech., 17, 1030 - 1032 (1999).
                                         [Full text from publisher]
                                         [Abstract from PubMed]
                                         [Comment published in Science (and its Pubmed link)]


Seraphin Lab TAP method

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Author: Séraphin@embl-heidelberg.de Last Modified, January 13, 2000