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Immunoprecipitation Protocol Immunoprecipitation protokol

Immunoprecipitation and protocols. Immunoprecipitation og protokoller.

Basic Immunoprecipitation Protocol IP: Grundlæggende Immunoprecipitation protokol IP:

1. Treat your cells Behandl dine celler

2. Harvest cells by adding 500 uL of solubilizing buffer, scrape and pass through syringe a 21-gauge needle X5 times to homogenize. Harvest celler ved at tilføje 500 uL af solubilizing buffer, skrabe og passerer gennem sprøjte en 21-gauge kanyle X5 gange for at homogenisere.

3. Conduct a TCA precipitation (this will be used to normalize if you radiolabeled the cells eg with S-35) Conduct en TCA nedbør (dette vil blive brugt til at normalisere hvis du radioaktivt cellerne fx med 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. Pre-clearing: Pre-klare ved at tilsætte 2 uL af X, mus eller kanin eller ged serum (hvor X er de dyrearter, hvori det primære antistof er blevet rejst) til hele prøver og inkubér i en rotator i 30 minutter ved stuetemperatur.

5. Remove X serum by incubating with 30 uL immunoprecipitin with 30 minutes rotation at room temperature. Fjern X serum ved Inkubation med 30 uL immunoprecipitin med 30 minutter rotation ved stuetemperatur.

6. Centrifuge samples for 3 minutes at 13, 000 rpm. Centrifuger prøverne i 3 minutter ved 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. Efter centrifugering, tilsættes 5 uL af primær antistof (X anti-Y særlige protein, hvor Y er den antistof rejst i et dyr, for din protein af renter) til supernatanter og inkuberes natten over ved 4 grader C.

8. Add 100 uL of immunoprecipitin to samples. Tilsæt 100 uL af immunoprecipitin til prøver. Alternatively you can use Zysorbin (Invitrogen). Alternativt kan du bruge Zysorbin (Invitrogen). (Zysorbin requires some minor changes to the protocol) (Zysorbin kræver nogle mindre ændringer til protokollen)

9. Centrifuge at 13, 000 rpm for 3 minutes. Der centrifugeres ved 13, 000 rpm i 3 minutter. Remove the supernatant. Fjern supernatanten.

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. Vask pellet 3 X med 1 ml koldt IP vaskebuffer (tilføj proteasehæmmere til wah buffer inden brug) med at ryste for 5-10 minutter mellem hver vask.

11. Use this pellet to run on a SDS-PAGE gel. Brug denne pillen til at køre på en SDS-PAGE gel. You can add SDS Sample Buffer (protein loading buffer) to this and load on gel directly after boiling. Du kan tilføje SDS Sample Buffer (protein loading buffer) til denne og belastningen på gel direkte efter kogning.

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