Labeling of the Target Protein

Photoaffinity labeling of the target protein for leaf-movement factor... 

Generally, it is difficult to detect synthesized proteins by CBB staining. To confirm the expression of the target protein, we usually perform fluorescent labeling of the in vitro translation products using the FluoroTect Labeling System (FluoroTect) (reeNote 22). 

The target molecule can be directly labeled with a fluorescent dye or it can be indirectly labeled via biotinylation and successive incubation with a streptaviclin, R-phycoerythrin conjugate. We recommend to biotinylate the target protein since it can be used both for MACS and for FACS then. For direct fluorescence labeling of the target molecule, NHS-coupled fluorescence dyes, e. g., Alexa Fluor 488 from Molecular Probes, can be used, following the procedure described in protocol 2. 

As shown in Figure 12.6, two types of assays are often used in analyzing possible protein interactions and evaluating protein expression levels direa and indirect (or sometimes called sandwich) assays. Direct assays typically involve a target protein that are prelinked onto an assay platform such as a well-plate array or a cover slip. Subsequently, detection of the target protein is carried out with a primary antibody that is labeled with a fluorochrome. On the other hand, more target-specific sandwich assays typically start with prelinking of an unlabeled primary antibody to... 

---