Unsticking

Related Articles Chasing Rainbows How to Maximize Immunofluorescence Multiplexing Background glow Cutting crossover Staying bright User: Xiaohu Gao, University of Washington, Seattle The project: Characterizing tumor tissue by analyzing as many as five coexpressed proteins in clinical tissue biopsies. The problem: Gao wanted to use quantum dots (Invitrogen) because they allow easier multiplexing, but dots pose two serious aggregation problems. First, each qu

Melissa Lee Phillips
Dec 1, 2007

User: Xiaohu Gao, University of Washington, Seattle

The project: Characterizing tumor tissue by analyzing as many as five coexpressed proteins in clinical tissue biopsies.

The problem: Gao wanted to use quantum dots (Invitrogen) because they allow easier multiplexing, but dots pose two serious aggregation problems. First, each quantum dot conjugates with a variable number of antibody molecules, so it can attach multiple proteins, possibly causing proteins to aggregate unnaturally and induce changes in cell signaling. Second, the dots themselves can form clumps in solution.

The solution: There's no way to guarantee that each quantum dot is bound to a single antibody, or even to measure how many antibodies a dot carries, says Gao, which means one can gauge relative but not absolute protein expression. It's best to ensure that the ratio of quantum dots to...