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Immunoprecipitation Protocol

Immunoprecipitation and protocols.

Basic Immunoprecipitation Protocol IP:

1. Treat your cells

2. Harvest cells by adding 500 uL of solubilizing buffer, scrape and pass through syringe a 21-gauge needle X5 times to homogenize.

3. Conduct a TCA precipitation (this will be used to normalize if you radiolabeled the cells e.g. with S-35)

4. Pre-clearing: Pre-clear by adding 2 uL of X, mouse or rabbit or goat serum (where X is the animal species in which the primary antibody was raised) to whole samples and incubate in a rotator for 30 minutes at room temperature.

5. Remove X serum by incubating with 30 uL immunoprecipitin with 30 minutes rotation at room temperature.

6. Centrifuge samples for 3 minutes at 13, 000 rpm.

7. After centrifugation, add 5 uL of primary antibody (X anti-Y specific protein, where Y is the antibody raised in an animal for your protein of interest) to the supernatants and incubate overnight at 4 degrees C.

8. Add 100 uL of immunoprecipitin to samples. Alternatively you can use Zysorbin (Invitrogen). (Zysorbin requires some minor changes to the protocol)

9. Centrifuge at 13, 000 rpm for 3 minutes. Remove the supernatant.

10. Wash pellet 3 X with 1 mL of cold IP wash buffer (add protease inhibitors to wah buffer prior to use) with shaking for 5-10 minutes between each wash.

11. Use this pellet to run on a SDS-PAGE gel. You can add SDS Sample Buffer (protein loading buffer) to this and load on gel directly after boiling.

©2012 Molecularstation.com


 

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