Table 1.

Troubleshooting Guide.

Problem	Possible Cause	Solution
Low signal for biotinylated proteins on western blot	Biotin ligase activity hindered by fusion to bait	Optimize linker length and flexibility; consider fusing the enzyme to a different terminus of the bait
	Low transfection efficiency	Ensure cells are low-passage, actively growing, and free of mycoplasma; optimize cell density at time of transfection
	Insufficient available biotin for robust labeling	Increase concentration of biotin in the cell culture medium
	Insufficient labeling time	Increase the duration of AirID fusion overexpression
	Poor blotting	Ensure blocking reagent is free of biotin; include commercially available biotinylated markers as a control for biotin detection; expect detection of endogenously biotinylated proteins of 72, 75, and 130 kDa
Bait activity significantly reduced on functional assay	Bait activity hindered by fusion with AirID	Optimize linker length and flexibility; consider fusing the enzyme to a different terminus of the bait
Low signal on immunofluorescence assay	Concentration of antibody (or streptavidin reagent) is too low	Optimize concentrations of antibodies and fluorophore-conjugated streptavidin
	Alteration of epitopes by organic solvent(s)	Fix cells in formaldehyde and permeabilize in Triton-X
Biotinylated protein signal in input and flow-through is largely unchanged	Too few beads were used	Use more beads (decrease the cell lysate:bead volume ratio)
	Free biotin saturates streptavidin resin to prevent capture of biotinylated proteins	Be sure cells and dishes are carefully washed with DPBS to remove free biotin present in culture media
High background banding on silver stain of eluate from biotin pull-down	Too many beads were used	Use fewer beads (increase the cell lysate:bead volume ratio)
	AirID overexpressed at too high a level	Increase the specificity during labelling by reducing the expression levels of AirID controls and fusions. This may require increasing the total number of cells used to acquire sufficient material.
Low signal on silver stain of eluate from biotin pull-down	Biotinylated proteins remain bound to the beads	Add a third elution step
	Too few beads were used	Use more beads (decrease the cell lysate:bead volume ratio)
	Not enough biotinylated proteins in the cell lysates	Scale up the proximity biotinylation experiment to harvest protein from more cells; express higher amounts of proteins during transfection