Coimmunoprecipitation (nuclear extracts)

1. Resuspend protein A Sepharose or protein G sepharose beads in IP BUFFER at 100 mg/ml

2. Swell beads by rotating 30 mins at RT. Wash 2x with IP BUFFER and resuspend at 100 mg/ml

3. For each IP reaction, mix 40 µl of 100 mg/ml protein A Sepharose with 2-20 µl antibodies in 400 µl IP BUFFER, containing protease inhibitors. Rotate over night at 4˚C.

4. Wash beads three times with IP BUFFER ( spin 2,000 rpm, 1 min and remove supernatant).

5. Transfer nuclear extract to tube containing washed antibody conjugate. Rotate at 4˚C, 2-4h.

6. Wash beads 8 times with 1 ml IP BUFFER (ice-cold) ( spin 1,000 rpm, 1 min and remove supernatant).

7. Resuspend in 10ul 6x protein sample buffer, heat for 5 min. at 95C, spin at 20 000g for 5 min. Move supernatant to new tube and use it for SDS PAGE.

IP BUFFER: 50 mM Tris-HCl pH7.5, 150 mM NaCl, 0.05% NP40.




Writing for MAPK resource
MAP kinase resource community
MAP Kinase Wiki
MAP Kinase Conference 2014

Latest MAP Kinase news

The Biochemistry, Biology and Pathology of MAP Kinases II, September 11-12, 2014, Vilnius, Lithuania

It`s our pleasure to invite you to the international event "The Biochemistry, Biology and Pathology of MAP Kinases II". The conference is organized by Enterprise Lithuania together with Swiss...

Next-Gen Kinase Inhibitors Conference - Cambridge Healthtech Institute, June 17 - 19, 2013

This established and well-recognized kinase conference addresses reoccurring questions about selectivity, safety, resistance and novelty. After many years of research and therapeutic developments of...

Control of the cell cycle progression by the MAPK Hog1

Eukaryotic cells coordinate various intracellular activities in response to environmental stresses, activating an adaptive program to maximize the probability of survival and proliferation. Cells...

Ultrasensitive MAPK/Erk activation in absence of protein synthesis in Xenopus oocytes

The mitogen-activated protein kinase (MAPK) cascade in Xenopus oocytes exhibits an all-or-none, ultrasensitive response, which is believed to result from a positive feed-back loop. Here we describe a...

Batsheva de Rothschild Seminar on Biochemistry, Biology and Pathology of MAP Kinases

This abstract book contains most of the abstracts presented to the Batsheva de Rothschild Seminar on Biochemistry, Biology and Pathology of MAP Kinases. An Aharon Katzir-Katchalski Meeting and an...


Project Sponsors & Friends


Swiss Institute of Bioinformatics Journalism differently - journalism about mass media

Lithuanian Biotechnology Association Enterprise Lithuania

PAGEPress Publications - scholarly open access journals International Society for Biocuration

brightstat.com

European Bioinformatics Institute

The Coins

DNASU

Ziedavimas
Cancers