Authors
Konstantinos Grintzalis, Ioannis Papapostolou, and Christos Georgiou
Summary
Several methods for protein determination have been developed [1] but the ones most commonly used today are based on the reaction of proteins with Commassie Brilliant Blue G-250 (CBB) [2] and alkaline Cu(II) [3]. The most recent modifications of these methods are the Sedmak assay [4] and the bicinchoninic acid (BCA) assay [5]. Another sensitive method for measuring proteins is the colloidal gold protein assay [6] which has been modified to quantify specific proteins of interest [7,8]. The present protocol is a very simple CBB-based protein assay which is more sensitive and less prone to interference than the commonly used Bradford, Sedmak and BCA assays [9]. It uses the hydrophobic reagents ammonium sulfate (AS) and trichloroacetic acid (TCA) to increase the binding of more CBB molecules per protein molecule (for mechanism see Figure 1). The protocol consists of three independent sub-protocols: general assay, microplate assay and microassay, with minimum detectable protein 100, 50 and 150 ng, respectively, which make it 100/40 and 200/5 fold more sensitive than the Sigma/Pierce Bradford and BCA assays, respectively [9].Further details
The protocol was published on Protocol Exchange on 27 October 2009. To see the entire protocol, click on the source link.Advertisement
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