- from PlantMethods.com. Click here for the full article.
Chromatin remodeling, histone modifications and other chromatin-related processes play a crucial role in gene regulation.
- 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.
- from the Farnham lab. Click here for full protocol link.
Day 1
1. Add formaldehyde directly to tissue culture media to a final concentration of 1%. We generally use 1 x 107 cells per antibody per timepoint. For cloning, multiple IPs are performed and pooled at later steps therefore we generally start with 1 x 108 cells. Incubate adherent cells on a shaking platform and suspension cells on a stir plate for 10 minutes at room temperature.
2. Stop the crosslinking reaction by adding glycine to a final concentration of 0.125 M. Continue to rock or spin at room temp for 5 minutes.
- 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.
- from Bart’s Lab. Click here for full link.
Includes information on:
RIPA buffer
NP40 buffer
PBS
TN
Boiling in SDS
Antisera
Bugs
Secondary Antibodies
Cell Lysis
Pre-clearing
Abbreviated protocol
Protocol from Hahn Lab. Click here for full protocol.
Chromatin IP Method
ChIP buffers
Notes on ChIP Method
Quantative PCR using 32P dCTP
METHODS:
Click here to link to the video
The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type .
0:11 Introduction11
0:27 Formaldehyde Crosslinking Cells27
2:22 Preparing Magnetic Beads142
4:13 Cell Sonication253
5:58 Chromatin Immunoprecipitation, Wash, and Elution358
8:53 Crosslink Reversal and DNA Purification533
9:58 Conclusion
Find a link to immunoprecipitation protocol with details and specifics, reagents and equipment.
- from eBioscience (click here for full protocol).
Immunoprecipitation involves the interaction between a protein and its specific antibody, the separation of these immune complexes with Protein G or Protein A, and the subsequent analysis by SDS-PAGE. This technique provides a rapid and simple means to separate a specific protein from whole cell lysates or culture supernatants.