Immunoprecipitation and Immune Complex Kinase Assay

- from the Bart’s Lab. Click here for full protocol link. 

1) In the cold room, wash cells with cold “Tris” (the same stuff used for TC).

Spin down the cells if you are working with suspension cells in a low speed swinging bucket centrifuge, resuspend in approximately 1 ml Tris and spin at 2K in a microfuge.

Wash on plate if adherent cells by aspirating medium, adding 2 to 10 ml Tris, and aspirating again.

Immunoprecipitation Protocol for Labeled Cells.

- from An Zhou, Johns Hopkins, 1995. Click here for full protocol. 

1. To your sample, add Super E, protease inhibitors, 1 mM cold Met, antibody.

TMT extract or medium - dilute with Super E to convenient volume; add protease inhibitors, cold Met and antibody.

Some antibodies work only after the antigen has been denatured (e.g. Ab 877, Ab 471): to 50 ul of sample in TMT, add 5 µl 10% SDS and incubate at 100 ^oC for 5 min; allow to cool to room temperature for 5 min; add IN THIS ORDER: 25 µl 15% NP-40, 180 µl Super E, 3 µl PMSF, 3 µl inhibitor mix, 10 µl antibody. Super E: 50 mm phosphate, 1% Triton.

Immunoprecipitation Method

Find a full immunoprecipitation protocol from the Hancock Lab. Click here for the full protocol link.

METHODS:

Immunoprecipitation Protocol Details

Find a link to immunoprecipitation protocol with details and specifics, reagents and equipment.

Full protocol link.

Cross Linking and Immunoprecipitation Protocol

- from Rockefeller Click here for link to full protocol.

General method for UV cross-linking of tissue/cell lines

Immunoprecipitation

CIP Treatment

3’ RNA Linker Ligation

PNK Treatment

SDS-PAGE and nitrocellulose transfer

RNA Isolation and Purification

Dephosphorylation

3’ RNA linker ligation

Methylation analysis by DNA immunoprecipitation.

Thu KL, Pikor LA, Kennett JY, Alvarez CE, Lam WL. J Cell Physiol. 2010 Mar;222(3):522-31.

Abstract

DNA methylation regulates gene expression primarily through modification of chromatin structure. Global methylation studies have revealed biologically relevant patterns of DNA methylation in the human genome affecting sequences such as gene promoters, gene bodies, and repetitive elements. Disruption of normal methylation patterns and subsequent gene expression changes have been observed in several diseases especially in human cancers. Immunoprecipitation (IP)-based methods to evaluate methylation status of DNA have been instrumental in such genome-wide methylation studies. This review describes techniques commonly used to identify and quantify methylated DNA with emphasis on IP based platforms. In an effort to consolidate the wealth of information and highlight critical aspects of methylated DNA analysis, sample considerations, experimental and bioinformatic approaches for analyzing genome-wide methylation profiles, and the benefit of integrating DNA methylation data with complementary dimensions of genomic data are discussed.