• Labeling

    • I would like to label 500 µg of antibody. What do you recommend?
    • Larger quantities of reagent can be labeled in two ways: 1) Parallel labeling of 100 µg of antibody/protein using Alexa Fluor® 647 Antibody Labeling Kit (A-20186) from Molecular Probes (Life Technologies). In this case, the labeled protein is pooled before measuring the absorbance and calculating protein concentration and degree of labeling. 2) Use Alexa Fluor® 647 Protein Labeling Kit (A20173) from Molecular Probes (Life Technologies), which is optimized for labeling of 1 mg of antibody.

    • My unlabeled reagent contains thimerosal or azide. Can I still label it?
    • Low concentrations of sodium azide (< 3 mM) or thimerosal (< 1 mM) will not significantly interfere with protein labeling. If concentrations are too high remove the sodium azide or thimerosal using a nanosep column (see the User Guide for more information).

    • How do I store/handle my reagents?
    • Capture reagent (biotinylated): Store in refrigerator (+4°C), or in freezer (- 20°C) for long-term storage. Always check the supplier’s product data sheet. Dilute in PBS-0.01% Tween (filtered) to working concentration. The recommended working concentration is 100 µg/ml. Detection reagent (Alexa-labeled): After labeling, dilute in PBS-0.2% BSA to 1000 nM. Store in aliquots (approx. 15 µl/vial) in freezer. Thaw a new vial at least every other week. Centrifuge detection reagent 12000 x g for 4 minutes. Dilute to working concentration in Rexxip F. The recommended working concentration is 50 – 5 nM. Always titrate this concentration for a new assay and also when performing a new labeling for an established assay.

    • How long are labeled reagents stable in the refrigerator?
    • Biotin-labeled reagents: several months. Alexa-labeled reagents: approximately 2-3 weeks. Alexa-labeled reagents should be diluted to 1000 nM in PBS, 0.2% BSA (Calbiochem) and aliquoted and at -20°C protected from light. Please remember to check the stability of the unlabeled reagent (data sheet).