The fl uorescent probe has been used extensively to label numerous proteins and other bio-
molecules, including actin (Glacy, 1983; Wang, 1985; Meige and Wang, 1986), myosin light
chains (Mittal et al., 1987), -actin (Simon and Taylor, 1988; Stickel and Wang, 1988), blood
coagulation factor Va (Isaacs et al., 1986), and histones (Murphy et al., 1982). The dye also
has been used to study conformational changes in proteins (Heuck et al., 2007), to study the
binding region of protein C on factor Va (Yegneswaran et al., 2007), and how fl avonoids affect
actin functions (Boehl et al., 2007). Hundreds of additional publications cite the use of this dye
for various biological detection applications.
The following protocol for labeling proteins with tetramethylrhodamine-5-(and-6)-iodoa-
cetamide represents a general guideline. The procedure should be optimized for each macro-
molecule being labeled to obtain the best F / P ratio to produce intense fl uorescence and high
activity in the fi nal complex.
Protocol
1. Prepare a 20 mM tetramethylrhodamine-5-(and-6)-iodoacetamide solution by dissolving
11.3 mg/ml of DMF. Prepare fresh and protect from light.
2. Dissolve the protein to be modifi ed at a concentration of 5–10 mg/ml in 50 mM sodium
phosphate, pH 7.5.
3. Slowly add 25–50 l of the tetramethylrhodamine-5-(and-6)-iodoacetamide solution to
each ml of the protein solution while mixing.
4. React for 2 hours at 4 ° C in the dark.
5. Remove excess reactant and reaction by-products by gel fi ltration using a desalting resin
or by dialysis.
Aldehyde/Ketone and Cytosine-Reactive Rhodamine Derivatives
Hydrazide groups can be coupled directly to aldehydes and ketones to form relatively stable
hydrazone linkages (Chapter 2, Section 5.1). Two rhodamine derivatives are commonly avail-
able that contain a sulfonyl hydrazine group off their No. 5 carbon on the lower-ring structure
(Invitrogen). They are based on the Lissamine and Texas Red structures and may be used to
label aldehyde- or ketone-containing molecules with an intensely fl uorescent probe. Although
most biomolecules don ’t contain aldehyde or ketone groups in their native state, carbohy-
drates, glycoproteins, RNA, and other molecules containing sugar residues (or diols) can be
oxidized with sodium periodate to produce reactive formyl groups. The use of modifi cation
reagents which generate aldehydes upon coupling to a molecule also can be used to produce a
hydrazide-reactive site (Chapter 1, Section 4.4).
DNA and RNA may be modifi ed with hydrazide-reactive probes by reacting their cyto-
sine residues with bisulfi te to form reactive sulfone intermediates. These derivatives undergo
transamination to couple with hydrazide- or amine-containing probes (Draper and Gold, 1980)
(Chapter 27, Section 2.1).
Lissamine Rhodamine B Sulfonyl Hydrazine
Lissamine rhodamine B sulfonyl hydrazine is a hydrazide derivative of sulforhodamine B that
can spontaneously react with aldehyde- or ketone-containing molecules to form a covalent,
2. Rhodamine Derivatives 427