AMCA-hydrazide

AMCA-Hydrazide 7-Amino-4-methylcoumarin-3-acetyl hydrazide MW 247. 1 Excitation = 345 nm Emission = 440-460 nm... 

Figure 9.26 AMCA-hydrazide can be used to label aldehyde-containing molecules, such as periodate-oxidized carbohydrates.

The hydrazide derivative of AMCA can be used to modify aldehyde- or ketone-containing molecules, including cytosine residues using the bisulfite activation procedure described in Chapter 27, Section 2.1. AMCA-hydrazide reacts with these target groups to form hydrazone bonds (Figure 9.26). Carbohydrates and glycoconjugates can be labeled specifically at their polysaccharide portion if the required aldehydes are first formed by periodate oxidation or another such method (Chapter 1, Section 4.4). 

AMCA-hydrazide has a maximal excitation wavelength of 345 nm and a maximum emission wavelength in the range of 440-460 nm. A solution of AMCA in PBS at a concentration of 16.7ng/ml (71.61 nmoles/ml) gives an absorbance at 345 nm of about 1.28. This translates into a molar extinction coefficient at this wavelength of about 13,900M em-1. Different solvents and conditions may alter this value somewhat. 

AMCA-hydrazide is soluble in DMSO or DMF and may be dissolved as a concentrated stock solution in either of these solvents prior to the addition of a small aliquot to an aqueous reaction medium. The solid and all solutions made from the fluorophore must be protected from light to avoid photo-decomposition. Prepare the stock solution fresh immediately before use. A suggested protocol on the use of this fluorescent probe may be obtained from the following method on the labeling of periodate-oxidized IgG. Optimization may be necessary to achieve the best level of fluorescent modification (F/P ratio) for a particular application. 

Dissolve AMCA-hydrazide in DMF at a concentration of 0.4 mg/ml. Protect from light. 

Add 50-100 pi of the AMCA-hydrazide stock solution to each ml of the oxidized antibody solution. 

Add 50-100 xl of the AMCA-hydrazide stock solution to each milliliter of the oxidized antibody solution. Note at a level of 50 p.1 probe addition, polyclonal human IgG will be modified at a level that gives an F/P ratio of about 0.113. Since the labeling occurs only at the oxidized carbohydrate sites, the fluo-rophore incorporation typically is less than that observed when using amine-reactive probes. 

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