Home › Immunology › Protein Complex Affinity Capture from Cryomilled Mammalian Cells
Immunology JoVE (Open Access) Citable · DOI

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

DOI: 10.3791/54518-v
What you'll learn
  • ✓ Perform cryogenic milling to disrupt mammalian cells while preserving protein complexes
  • ✓ Generate cell extracts from cryomilled cell powder for downstream analysis
  • ✓ Apply affinity capture using antibody-coupled paramagnetic beads to isolate target protein complexes
Protocol

Biopharma Insights Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Difficulty
advanced
Total time
~4–6 hours per sample batch (including cryomilling, extract preparation, and affinity capture)
Model organism
Mammalian cells (unspecified cell line)
Biosafety
BSL-1

Steps

1
Harvest and freeze mammalian cells

Collect cultured mammalian cells and freeze them rapidly to preserve protein complex integrity before cryogenic disruption.

▶ 00:30
2
Perform cryogenic milling of frozen cells

Disrupt frozen cell samples using solid-state milling at cryogenic temperature to generate a fine cell powder while maintaining native protein interactions.

▶ 03:09
3
Prepare cell extract from milled powder

Resuspend and lyse the cryomilled cell powder in extraction buffer to produce a soluble cell extract containing protein complexes.

▶ 04:50
4
Bind protein complexes to antibody-coupled beads

Incubate cell extract with paramagnetic beads conjugated to affinity antibodies to capture target protein complexes from the extract.

▶ 06:02
5
Isolate and recover affinity-captured complexes

Use magnetic separation to isolate antibody-bead complexes from the extract, then elute and recover captured protein complexes for downstream analysis.

▶ 07:41
6
Evaluate affinity capture optimization parameters

Compare results across different experimental conditions to assess factors affecting capture efficiency and complex recovery yield.

▶ 09:38
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