The IP-FCM method is presented, which allows a sensitive, robust, biochemical assessment of native protein-protein interactions, without requiring genetic engineering or large sample sizes.
Total time
~4–6 hours per sample (including incubation steps)
Steps
1
Introduce IP-FCM method and workflow
Overview of the immunoprecipitation detected by flow cytometry (IP-FCM) technique for assessing native protein-protein interactions without genetic engineering.
▶ 00:58
2
Couple monoclonal antibody to magnetic beads
Conjugate primary monoclonal antibodies to magnetic beads to prepare immunocapture reagents for subsequent immunoprecipitation.
▶ 01:31
3
Prepare lysate and perform immunoprecipitation
Generate post-nuclear lysate from cells and incubate with antibody-coupled beads to capture target protein complexes.
▶ 04:37
4
Probe bead-captured protein with detection antibodies
Incubate immunoprecipitated beads with fluorescently-labeled secondary antibodies to detect associated proteins.
▶ 07:07
5
Acquire data by flow cytometry
Run bead-bound samples through flow cytometer to quantify fluorescent signal and identify protein-protein interactions.
▶ 09:11
6
Interpret representative flow cytometry histogram
Analyze and visualize flow cytometry output to distinguish positive IP signal from background.
▶ 11:02