Tnf α protein

Commercially acquired preparations of MMP-9, TNF-α, IL1-α and IL1-β proteins and antibodies were used as received. 4-ATP and 2-NT were used as the Raman active molecules in this study to estimate the performance of the fabricated substrate. Representative SERS spectra of the Raman reporter molecules were shown in Figure S1. TNF-α protein (10 µg/mL, Abcam) was modified with 14 mM Traut’s reagent and filtered using an Ultra-0.5 Centrifugal Filter Unit (Amicon) to remove excess Traut’s reagent. EDC (400 µL of 50 mM) was added to 100 µL of 1 mg/mL anti-TNF-α solution and incubated for 5 min. NHS (400 µL of 10 mM) was then added to the mixture and left to incubate for 5 min. Next, 100 µL of 1 mM 4-ATP was added to the EDC-NHS anti-TNF-α solution and left to incubate for 30 mins. The solution was subsequently filtered to remove excess unreacted reagents and the resultant protein filtrate was reconstituted to 0.1 mg/mL anti-TNF-α modified with 4-ATP (anti-TNF-α-4-ATP).