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

Journal: bioRxiv

Article Title: NAADP elicits two-pore channel currents by lifting Lsm12-mediated inhibition of PI(3,5)P 2 activation

doi: 10.64898/2026.04.13.718294

Figure Lengend Snippet: (A) Representative time course of TPC2 PM currents at −120 mV during perfusion with 0.3 µM PI(3,5)P 2 . After current activation under control conditions, 0.5 µM Lsm12 was applied to inhibit the current, and subsequent washout of Lsm12 led to recovery. (B, C) Summary of the normalized current amplitudes derived from the maximal (activation) and minimal current (inhibition) levels ( B ) and time constants (τ) ( C ) during the control, Lsm12 wash-on, and wash-off phases from recordings as shown in A ( n = 5). (D) Effects of heat-treated Lsm12 on PI(3,5)P 2 -activated TPC2 PM currents, normalized to currents recorded in the absence of heat-treated Lsm12. (E) Fraction of unbound BODIPY FL PI(3,5)P 2 remaining after incubation of 0.3 µM lipid with 0.05, 0.3, or 1 µM 6×His-tagged Lsm12 ( n = 4), determined from the fluorescence remaining in solution after precipitation of bead-bound Lsm12 with IMAC beads. (F) Representative I–V relationships of constitutively active TPC2 PM carrying the L690A/L694A mutation, recorded under bath conditions alone or after addition of the indicated concentrations of Lsm12 or PI(3,5)P 2 . (G, H) Representative I–V relationships of TPC2 PM currents activated by 0.3 µM ( G ) or 1 µM ( H ) PI(3,5)P 2 in the absence and presence of the indicated concentrations of Lsm12. (I) Concentration-response relationships for inhibition by Lsm12 of TPC2 PM currents activated by 0.3 µM ( n = 10) or 1 µM ( n = 9) PI(3,5)P 2 . (J) PI(3,5)P 2 concentration-response relationships for TPC2 PM currents in the absence and presence of 2 µM Lsm12. (K) Lsm12 concentration-response relationships for TPC2 PM pseudo-WT ( n = 10) and Δ45–50 mutant ( n = 8) channels activated by 0.3 µM PI(3,5)P 2 . Left, modeled structure of Lsm12 highlighting residues 45–50 in stick representation. Summarized data are presented as plots showing mean ± SEM, with or without individual data points, as indicated. ns, not significant ( P > 0.05).

Article Snippet: 20 μl Nuvia IMAC (immobilized metal affinity chromatography) Ni-charged resin (Bio-Rad #7800800) pre-equilibrated with the K + -based solution was added and incubated for 10 min. After centrifugation to pellet the resin, the fluorescence of the unbound BODIPY FL PI(3,5)P 2 in the supernatant was measured by a plate reader.

Techniques: Activation Assay, Control, Derivative Assay, Inhibition, Incubation, Fluorescence, Mutagenesis, Concentration Assay