This is a cached page for the URL ( To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
The Science Advisory Board - Protocols, Product Reviews, Member Forum, and Science News
Note: You are seeing this message either because your browser has not loaded our stylesheets, or because your browser does not support stylesheets (CSS). Please upgrade to a relatively modern browser to improve your experience. Not sure what to upgrade to? Try Firefox.
The Science Advisory Board
Screen Name: 


Welcome to The Science Advisory Board's extensive database of research protocols for the biomedical sciences. Protocols are organized by techniques and are fully searchable by key word(s). Protocols are divided into a brief description of the methods, a step-by-step overview of the procedure, a collection of recipes, the supplies needed, and any helpful tips. Members submitting protocols will be rewarded through the ViewPoints system.
... back to articles list

Preparation of cytoplasmic extracts for the application in a cell-free system

Posted on Monday, November 24, 2003

Cells are grown to 80% confluency, then harvested, washed and disrupted in KPM buffer by freezing-thawing cycles with liquid nitrogen essentially as described by Fearnhead et al., 1997, Genes Dev. 11: 1266-76.

Grow cells in a 160 cm2 flask (25 ml medium) up to about 80% cell density.
Remove the RPMI medium and detach cells by incubating with 10 ml trypsin/EDTA (Sigma) for 5 min at 37C
For the inactivation of trypsin, add 10 ml of Bovine Calf Serum (BCS) to the trypsinized detached cells. Combine harvested cells with RPMI which was previously removed and spin at 200xg for 5 min.
Wash cells once with 5 ml of fresh RPMI medium.
Wash cells once with 5 ml icecold PBS.
Wash cells once with 1 ml icecold KPM (without Cytochalasin B).
Resuspend cells in about 1 volume (same volume as cell pellet) of KPM (containing 10 g/ml Cytochalasin B).
Lyse the cells by freezing the cell-suspension in liquid nitrogen and successively thawing them in a warm water bath. Repeat three times.
Spin lysate at 16000 g (14000 rpm in Eppendorf microfuge) for 20 min at 4C
The supernatant is the cytoplasmic extract that can be used in cell-free experiments. If necessary, the supernatant can be spun a second time to remove residual membrane fragments.
Measure protein content (will be approximately 15 g/l) and store extracts at -70C in aliquots until required (up to several weeks).

KPM BUFFER also called Extract Preparation Buffer (EPB):

RECIPE for 5 ml:

50 mM PIPES (pH 7.0)
50 mM KCl
2 mM MgCl2
1 mM DTT
0.1 mM PMSF
2 g/ml Leupeptin
2 g/ml Pepstatin
2 g/ml Aprotinin
with or without 10 g/ml Cytochalasin B
2.5 ml of 100 mM PIPES, pH 7.0 incl.
250 l of 1 M KCl
100 l of 100 mM MgCl
5 l of 1 M DTT
50 l of 10 mM PMSF
10 l of 1 mg/ml Leupeptin
10 l of 1 mg/ml Pepstatin
10 l of 1 mg/ml Aprotinin
2.04 ml H2O nuclease free



Submitted by: manny440

Scientific & Medical
Experts Needed!

The Science Advisory Board is the world's most established network of life scientists!

Voice your opinions on companies, products, protocols and even humor in a lively, real-time, interactive Online Community of over 42,000 life science & medical professionals.

Redeem generous rewards for participation in studies, contributing website content and referring colleagues.

Join Right Now!
(It's Free!)

Search This Site
only search
only search Forums
What's this?