Biotin-Celastrol Affinity Purification

Neuron lysates were incubated with biotin, celastrol‐biotin, or a combination of celastrol‐biotin and celastrol at 4 °C overnight. The lysates were subsequently subjected to pull‐down using streptavidin‐conjugated beads (Softlink soft release Avidin Resin, PROMEGA) at 4 °C for an additional 4 h. After extensively washing with PBS, the beads were boiled in 2 × loading buffer, and the supernatants were used for western blot analysis of EPAC‐1.

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Li X S.r., Liu W., Jiang G., Lian J., Zhong Y., Zhou J., Li H., Xu X., Liu Y., Cao C., Tao J., Cheng J., Zhang J.H, & Chen G. (2024). Celastrol Ameliorates Neuronal Mitochondrial Dysfunction Induced by Intracerebral Hemorrhage via Targeting cAMP‐Activated Exchange Protein‐1. Advanced Science, 11(19), 2307556.

Publication 2024

Softlink soft release Avidin Resin

Corresponding Organization : First Affiliated Hospital of Soochow University

Other organizations : Nanjing University, Loma Linda University

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Variable analysis

independent variables

Biotin
Celastrol-biotin
Combination of celastrol-biotin and celastrol

dependent variables

EPAC-1 expression

control variables

Incubation temperature (4 °C)
Incubation duration (overnight for lysate incubation, 4 h for pull-down)
Washing buffer (PBS)

controls

Positive control: Not specified
Negative control: Not specified

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