profinity imac resin  (Bio-Rad)

Journal: bioRxiv

Article Title: Structural divergence in N-terminal domains of AAA proteases paraplegin (SPG7) and FtsH indicates a key structural function in complex formation

doi: 10.64898/2026.04.22.720153

Figure Lengend Snippet: (A) IMAC purification of refolded, un-cleaved Para-IMS, showing on SDS-PAGE flow through (FT), wash (W), elution fractions of increasing imidazole concentration. His6-tagged Para-IMS is at an approximate size of 14k Da. (B) Size exclusion chromatography and accompanying SDS-PAGE after TEV-cleavage showing His6-Para-IMS as peak 1, just below 15 kDa on the SDS-PAGE, and cleaved Para-IMS (peak 2) at 10 kDa. Both hydrogenated and deuterated proteins showed similar profiles in IMAC and gel filtration. (C) CD wavelength spectrum at 20 °C (solid line) and 90 °C (dashed line) confirming a folded paraplegin-IMS protein. (D) Thermal denaturation of paraplegin-IMS protein and sigmoidal fit to determine the melting temperature T m at 208 nm with 48 °C. Shown are averages and standard deviations for three experiments. ( E) SDS-PAGE of IMAC purification of His 6 -FtsH (20-97) with reference protein lysozyme in lane 1 (M), flow through (FT) and elution fractions in following lanes labelled by their fraction numbers. (F) SDS-PAGE from anion exchange chromatography with the reference proteins lysozyme and annexin A1 in lane 1 (M), sample (S), flow through (FT) and wash (W) and elution fractions labelled by their fraction numbers in following lanes. (G) Size exclusion chromatography in conjunction with light-scattering analysis reveal a peak 1 at around 26 kDa to 30 kDa with a shoulder as peak 2 around 15 kDa. Elution profiles of known gel filtration standard proteins (BioRad) known marker proteins shown for comparison. (H) CD wavelength spectrum of FtsH-IMS showing folded protein. (I) Thermal denaturation of FtsH-IMS region at 222 nm displays a two-state transition curve with a melting temperature T m of 49 °C. Shown is the average of three independent experiments and their standard deviation as error bars with fit as solid line.

Article Snippet: Para-IMS was purified from lysate after high-speed centrifugation to remove unbroken cells using Profinity IMAC resin (#156-0123, Bio-Rad) and eluted using an imidazole gradient consisting of buffer A (50 mM Na phosphate buffer pH 7.5, 200 mM NaCl, 10 mM imidazole) and buffer B ( 50 mM Na phosphate buffer pH 7.5, 200 mM NaCl, 250 mM imidazole).

Techniques: Purification, SDS Page, Concentration Assay, Size-exclusion Chromatography, Filtration, Chromatography, Marker, Comparison, Standard Deviation