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64 lines
1.9 KiB
Markdown
64 lines
1.9 KiB
Markdown
---
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name: Gentle Co-Immunoprecipitation Protocol
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id: PROT-0012
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version: 1.0
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description: A gentle co-immunoprecipitation protocol for preserving protein-protein interactions
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author: JM Jordan
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created: 2023-01-01
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materials:
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- Protein lysate
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- RIPA buffer with protease inhibitor tablets
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- Antibody (2 µg per reaction)
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- Beads (50% slurry)
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- PBS with 0.02% Tween
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- Non-denaturing loading buffer (1X)
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- LoBind tubes
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- Liquid nitrogen
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steps:
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- "Prepare protein lysate (3600 µg protein)"
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- "Preclear with beads"
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- "Add antibody and incubate overnight at 4°C"
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- "Add washed beads and incubate"
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- "Wash beads with PBS-Tween"
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- "Elute proteins with non-denaturing buffer"
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- "Store samples at -80°C"
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notes: |
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This is a gentle protocol designed to preserve protein-protein interactions
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Uses minimal washing steps without extended rotations
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Includes preclearance step to reduce non-specific binding
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---
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#Protocol
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by JM Jordan 2023
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1. Prepared a lysate solution with 3600 ug of protein for HFA and HFB
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2. Adjusted to 1mL each with RIPA+Tablets
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3. Precleared solution by adding 50ul of 50% beads to each tube of lysate
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4. Aliquoted into 3 tubes/lysate (6 tubes total) and adjusted each to 1mL with RIPA+Tablets
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5. Added 2 ug of antibody to each tube
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6. Rotated overnight at 4C
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7. RT rotation for 1h
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8. Washed 600ul beads with PBS + 0.02% Tween
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9. Resuspended beads in 600ul RIPA+Tabs
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10. Added 100ul of bead solution to each tube
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11. Rotated at RT for 1h
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12. Washed beads 3X with 1ml PBS + 0.02% Tween with gentle pipet mixing (no 5 min rotation)
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13. After final wash, resuspended beads in 200 ul PBS + 0.02% Tween and transferred to fresh LoBind tubes
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14. Added 30 ul 1X Non denaturing loading buffer and eluted as usual at 37C for 5 min
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15. Separated solution from beads into new LoBind tubes and snap froze in LN2 and stored at -80C.
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16. Note: Freeze Input and leftover |