Recovery from ITC reactions

Nickel Beads Protocol

Based on Ejje, Soe, Gu, and Codd (2013) Metallomics in which they prep XAD-2 samples from extract for iron binding and assays. 

Braich and Codd, (2008) Analyst. In which they show dfoB binds to a Ni2+ column

Protocol

• Obtain 50 uL of Nickel bead slurry (check capacity?)
  • The binding capacity is typically greater than or equal to 7.5 mg His-tagged fusion protein / 1 mL bed volume.
• Wash with 5 CV of 16 ohm water (milliQ water)
• 5 CV 20 mM Hepes pH 7.5 to prepare*
• bind 50 - 300 uL product mix from ITC reactions (2mM) and incubate with gentle shaking for 5 minutes at RT (keep the flowthrough). Product mix should be spun down in a centrifuge for 2 minutes at 11,000 g to remove protein ppt. 
• wash with 5 CV (250 uL) of Hepes pH 7.5 (keep this and test)
• elute with 1-2 CV 20 mM FeCl3 in Hepes, decant supernatant, then 50 uL Hepes twice, added to the elution tube. 
• wash beads with milliQ water and store. 

 

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• note: you might also choose to strip the column with 0.5 CV 100 mM EDTA, 20 mM phosphate buffer pH 7.2, 300 mM NaCl, then clean with 5 CV phosphate buffer, 5 CV of milliQ water. Reload with Ni2+ using 0.5 CV of 0.1 M NiSo4 * 6H2O followed by 5 CV milliQ water, 5 CV phosphate buffer, 1 CV imidazole buffer to remove excess Ni2+ and then equilibrate with 3 CV binding buffer. 

Quantifying dfos using UV/Vis

dfos will absorb well at 435 nm if bound to Fe3+. Excess free iron will absorb at 340 nm. Nucleotides like AMP, ATP and the adenylate intermediate will absorb at 260 nm and our DesD protein absorbs at 280 nm. 

Standard curve in triplicate for dfoG bound to Fe(III) at 435 and 487 nm.
DfoG Iron(III) complexation standard curve.xlsx
Signal at 435 nm was within the detector range for the entirety of the curve making it the best option for monitering when recycling substrate. The equation from Linest analysis is abs 435 =0.98023x+0.03361(x is dfoG between 0.05 - 1.5 mM). 

Separating dfos on Reverse Phase HPLC

Chelate dfos with freshly prepared FeCl₃ in acetonitrile (orange solution, peak abs 340 nm), in a 1:10 ratio. Load onto a 4 mm x 250 mM Prontosil column in 8% Acetonitrile, 0.5% acetic acid running buffer. Wash and run at 1 mL/min will elute dfos according to the table below (run at 0.5 mL to better separate but expect double retention time): 

analyte	retention time (min)	wavelength of trace (nm)
HSC	3.4'	435 nm
adenylate intermediate	3.8'	260 nm (adenylate) and 435 nm (dfo)
dfoB	7 '	435 nm
dfoG	8'	435 nm
dfoE	17'	435 nm
ATP/AMP	2.4' (void volume)	260 nm

Under chelated iron can result in a trailing shoulder peak for any dfo ~30s-1 minute past the main peak.