Oct 22, 2025
In Situ Pull-down Protein Interaction Analysis
- Katja Piltti1,
- Zeina Elrachid1
- 1University of California, Irvine
Protocol Citation: Katja Piltti, Zeina Elrachid 2025. In Situ Pull-down Protein Interaction Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx4qjzl8j/v1
Manuscript citation:
Piltti KM et al. C1q drives neural stem cell quiescence by regulating cell cycle and metabolism through BAI1. Nature Communications (In press)
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 21, 2025
Last Modified: October 22, 2025
Protocol Integer ID: 230356
Keywords: In situ pull-down assay, Protein–protein interaction assay, Magnetic bead pull-down, Affinity purification assay, Protein complex analysis, His-tag pull-down, Recombinant protein binding assay, Magnetic bead protein capture, Neural stem cells, Stem cell biology, protein interaction analysis an in situ, protein interaction analysis an, direct interactions between c1q, direct binding of c1q, bai1, c1q, direct binding, direct interaction
Funders Acknowledgements:
Wings for Life
Grant ID: WFL-US-01/18
Abstract
An in situ His-tag pull-down assay to test direct interactions between C1q, BAI1, and p32 demonstrates direct binding of C1q–BAI1, C1q–p32, and BAI1–p32, and that C1q-BAI1-p32 can be pulled down as a complex.
Materials
- His-tag recombinant human BAI1 (SinoBiological, 4969-BA)
- His-tag p32 (SinoBiological, 11874-H08E)
- Magnetic beads (Invitrogen, 10103D)
- Purified human C1q (MyBioSource, MBS147305)
- Recombinant untagged human p32 (LSBio, LS-G3375-20)
- Binding/wash buffer (50 mM sodium phosphate, 300 mM NaCl, 0.01% Tween-20)
- Pulldown buffer (3.25 mM sodium phosphate, 70 mM NaCl, 0.01% Tween-20)
- His-elution buffer (300 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, 0.005% Tween-20)
Troubleshooting
Prepare Bait Proteins
Dissolve 5 µg of His-tag recombinant human BAI1 (SinoBiological, 4969-BA) and His-tag p32 (SinoBiological, 11874-H08E) in 1x binding/wash buffer (50 mM sodium phosphate, 300 mM NaCl, 0.01% Tween-20).
For each pull-down, immobilize the bait proteins on 2 mg of magnetic beads (Invitrogen, 10103D) in Eppendorf tube(s) by incubating on a rocker for 10 minutes at room temperature (RT).
Collect Excess Bait
Place tubes on a magnet (Invitrogen, 12321D) to separate beads from the buffer.
Collect the buffer containing excess bait for Western blot validation.
Wash Beads
Wash the beads four times with 1x binding/wash buffer to remove any remaining excess bait protein.
Prepare Prey Proteins
Dissolve 5 µg of purified human C1q (MyBioSource, MBS147305) and recombinant untagged human p32 (LSBio, LS-G3375-20) in 1x pulldown buffer (3.25 mM sodium phosphate, 70 mM NaCl, 0.01% Tween-20).
Incubate Prey with Bait
Incubate the single prey proteins or combination of the prey proteins with the magnetic bead-immobilized bait in the Eppendorf tube(s) on a rocker for 15 minutes at RT.
Capture Protein Complexes
Use a magnet to separate the beads and wash four times with 1x binding/wash buffer to remove unbound prey proteins.
Elute Proteins
Elute the bound prey and bait using his-elution buffer (300 mM imidazole, 50 mM sodium phosphate, 300 mM NaCl, 0.005% Tween-20).
Analyze the eluted samples using Western blotting to confirm interactions.