Site-specific labeling of affinity tags in fusion proteins

A staining solution comprising

• a) a fluorescent compound comprising nickel ions, wherein the fluorescent compound has the formula A(L)m(B)n wherein A is a fluorophore that is xanthene, coumarin, cyanine, acridine, anthracene, benzofuran, indole or borapolyazaindacene, L is a linker, B is an acetic acid binding domain that is NTA or BAPTA, m is an integer from 1 to 4 and n is an integer from 1 to 6;
• and,
• b) a buffer having a pH of about 7.0 to about 9.0 comprising about 20 mM phosphate;
• with the proviso that the binding domain does not comprise an antibody or fragment thereof.

A method for detecting a poly-histidine affinity tag containing fusion protein in a sample, the method comprising:

• i) immobilizing the sample on a solid or semi-solid matrix to prepare an immobilized sample;
• ii) contacting the immobilized sample with a staining solution to prepare a stained sample, wherein the staining solution comprises
• a) a fluorescent compound having formula A(L)m(B)n wherein A is a fluorophore, L is a linker, B is an acetic acid binding domain capable of selectively binding to a poly-histidine affinity tag, m is an integer from 1 to 4 and n is an integer from 1 to 6;
• and,
• b) a buffer having a pH of about 7.0 to about 9.0;
• iii) incubating the stained sample for a sufficient amount of time to allow the fluorescent compound to associate with the poly-histidine affinity tag to prepare an incubated sample;
• iv) illuminating the incubated sample with a suitable light source to prepare an illuminated sample;
• and
• v) observing the illuminated sample whereby the fusion protein is detected.

A kit for detecting an affinity tag containing fusion protein, wherein the kit comprises;

• a staining solution comprising:
• a) a fluorescent compound having formula A(L)m(B)n wherein A is a fluorophore, L is a linker, B is an acetic acid binding domain capable of selectively binding to a poly-histidine affinity tag, m is an integer from 1 to 4 and n is an integer from 1 to 6;
• and,
• b) a buffer having a pH of about 7.0 to about 9.0;
• with the proviso that the binding domain does not comprise an antibody or fragment thereof.