Identification of NLS

The sequence comprising the NLS must be both necessary and sufficient to cause translocation to the nucleus.

1.  Identify regions of the protein that have a high probability of being an NLS sequence.

2.  Perform mutagenesis and alter or delete these residues

3.  Define which deletions destroy the ability of the protein to localize to the nucleus.

These amino acids are necessary for nuclear import.

4.  Add the amino acids necessary to import to a protein that is normally not imported to the nucleus.  For example, GFP could be used.

5.  Monitor cells for import of the protein to the nucleus.  If the added amino acids confer the ability to be transported, then this sequence is sufficient to be an NLS.

(Rodriguez-Vilarrupla et al. 2002)