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Protocol
Immunoprecipitation
Keywords: immunoprecipitation, ripa, nonidet, tris
Immunoprecipitation protocol for separation of proteins from whole-cell lysate.
Immunoprecipitation is a fast and simple method to separate specific proteins from whole-cell lysate. The technique is based on the interaction between a protein and its specific antibody and the result is subsequently analyzed by SDS-PAGE. Immunoprecipitation can be used to confirm the identity or study biochemical characteristics, post-translational modifications, and expression levels of a protein of interest.
Protocol is that of ROCHE anti-His6 mouse monoclonal instructions.
Required Materials
- Eppendorf tubes
- Pipette tips
- Pipette
- Gelatin
- Secondary antibody , 2 hours swine anti-rabbit HRP conjugated (Dako) 1:3000 in 0.5 % BSA
- Primary antibody , 2 hours abcam Phosphoserine Rabbit polyclonal (ab9332) in 0.5 % BSA. 3 µg/ml
- BSA
- Proteine G Agarose
- Centrifuge
- SDS-gel
Main steps
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Lyse cells with cold lysis buffer (RIPA buffer or other) for 30 minutes…
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Pellet the beads at 12.000 g for 1 minute and transfer supernatant to a…
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Add mouse anti-his antibody (5 µg/ml). Incubate for at least 3 hours,…
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Collect the immuno-complex by adding 50 µl protein G agarose (ROCHE) and…
- (1GK6YRX)
- (1GK7EXB)
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Resuspend in loading buffer. Boil by heating to denature. Load into…
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Blocking with 3% BSA and 0.1 % Gelatin. o/n.
Instructions
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Lyse cells with cold lysis buffer (RIPA buffer or other) for 30 minutes at 4°C rotating or on ice. Centrifuge for 5 minutes at maximum speed with a volume around 106 cells/ml lysis buffer (150 µl) Transfer the supernatant to a new tube and add 50µl of protein G agarose. Incubate for at least 3 hours or overnight at 4°C.
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Pellet the beads at 12.000 g for 1 minute and transfer supernatant to a fresh tube.
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Add mouse anti-his antibody (5 µg/ml). Incubate for at least 3 hours, preferably overnight, at 4°C.
-
Collect the immuno-complex by adding 50 µl protein G agarose (ROCHE) and incubate at least 3 hours or overnight.
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Collect the complexes (agarose-antibody-antigen) by centrifugation for 1 minute at 12.000 g.
Discard the supernatant and resuspend the beads in 1 ml high salt buffer and incubate 20 minutes at 4°C.
Repeat step.
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Collect the complexes (agarose-antibody-antigen) by centrifugation 1 minute at 12.000 g.
Discard the supernatant and resuspend the beads in 1 ml low salt buffer and incubate 20 minutes at 4°C.
Repeat step.
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Resuspend in loading buffer. Boil by heating to denature. Load into SDS gel.
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Blocking with 3% BSA and 0.1 % Gelatin. o/n.
