Nickel-nitrilotriacetic acid resin pull down assay is an affinity chromatography procedure that uses a bait protein, P. lutzii GAPDH in our case, immobilized and incubated with a protein source containing putative protein preys. The assay was performed in native conditions and the results included direct and indirect associations between bait and prey proteins. Firstly, recombinant GAPDH was immobilized onto Ni-NTA resin following the purification assay without the elution step. Then, 300 μg of P. lutzii cell lysate containing the protein extract was incubated for 3 h on ice and under gentle agitation. Next, the column containing bait and prey proteins was washed with native wash buffer 5 times to reduce unspecific interactions or contaminants and then, the complex bait-preys was eluted with native elution buffer. The eluted sample underwent tryptic digestion and the digested peptides were separated further via NanoUPLC-MSE and analyzed using a nanoACQUITY system (Waters Corporation, Milford, Manchester, United Kingdom) in order to identify the proteins that possibly interacted with GAPDH.
A control sample was prepared similarly but 300 μg of P. lutzii protein extract was incubated with Ni-NTA without the immobilized GAPDH and the proteins identified in both experiments were excluded from the results.
A control sample was prepared similarly but 300 μg of P. lutzii protein extract was incubated with Ni-NTA without the immobilized GAPDH and the proteins identified in both experiments were excluded from the results.