[19] Quantifying amyloid by congo red spectral shift assay

The specific protocols discussed in this chapter refer to the use of the CR spectral shift assay to quantify Aβ aggregation. However, this method has the potential to be very general and the strategy for optimizing the method for other amyloid proteins is later discussed in detail. The CR spectral shift assay has several advantages over other approaches. First, this method requires only a simple spectrophotometer, making it broadly applicable. Second, it requires no radioisotopes or other expensive reagents. It is rapid and technically simple because there is no need to separate amyloid-bound from free indicator. Perhaps the greatest advantage of this technique is its ability to quantitate dye–amyloid interactions in absolute terms, allowing the direct comparison of results from day to day and from one laboratory to another. Initial discussion of the mathematical derivation of the equations used in this method incurs the risk of obscuring the relative ease and straightforward nature of this technique. To avoid this, this chapter first presents a basic protocol that allows rapid and direct application of the assay to the quantitation of Aβ fibril test samples prior to discussion of the theoretical basis of the method.