Chapter 7 – Complementary Experimental Tools  297

A smaller length scale version of this approach has been recently developed called “single-​

molecule pull-​down” or “SimPull” (Jain, 2011), which is a memorable acronym to describe

a range of surface-​immobilization assays developed by several research groups using fluores­

cence microscopy to identify different proteins present in a solution from their capacity

to bind to target molecules conjugated to a microscope slide or coverslip (Figure 7.6a). For

example, the coverslip surface is conjugated first with a reagent such a PEG–​biotin, which

both serve to block the surface against nonspecific interactions with the glass from subse­

quent reagents used, and the flow cell is then subjected to a series of washes and incubation

steps, first to flow in streptavidin/​NeutrAvidin that will then bind to the biotin (see the pre­

vious section of this chapter). Biotinylated antibody is then flowed in, which can bind to the

free sites on the streptavidin/​NeutrAvidin (as discussed earlier, there are four available sites

per streptavidin/​NeutrAvidin molecule), which are not bound to the biotin attached to the

PEG molecules.

This antibody has been designed to have binding specificity to a particular biomolecule

to be identified from the cell lysate extract, which is then flowed in. This single-​molecule

prey protein can then be identified using immunofluorescence using a fluorescently labeled

secondary antibody that binds to the Fc region of biotinylated primary antibody (see the pre­

vious section of this chapter) or directly if the prey protein has been tagged previously using a

fluorescent protein marker. TIRF microscopy (see Chapter 3) can then be used to identify the

positions and surface density of bound prey protein. Additional methods involving stepwise

FIGURE 7.6  High-​throughput protein detection. (a) Single-​molecule pull-​down, which uses

typically immunofluorescence detection combined with TIRF excitation. (b) Yeast two-​hybrid

assay; an activation transcription factor is typically composed of binding domain (BD) and acti­

vation domain (AD) subunits (left panel). BD is fused to a bait protein and AD to a prey protein

(middle panel). If bait and prey interact, then the reporter gene is expressed (right panel).