Dr. Sebastian Auer, Lab Ibañez-Tallon
Max-Delbrück Center for Molecular Medicine, Berlin-Buch, Germany
March 2011
Protein extraction from mammalian cells
 Wash cells twice with 1x PBS
 Scrape off cells in 1ml 1x PBS, and collect in 1.5ml tube (1x10 cm dish per sample)
 Pellet cells (1000 rpm, 2 min), discard SN and resuspend in 1ml ice-cold Resuspension Buffer
 All following steps are carried out at 4°C
 Homogenize and lyse cells by passing several times through a 27G needle (grey ones)
 Centrifuge (13000 rpm, 10 min, 4°C)
 Collect SN (cytosolic fraction), shock freeze (liquid N2), store at -80°C
 Resuspend pellet (membrane fraction) in 500 µl Ripa buffer for membrane protein extraction
 Incubate on shaker (15-30 min, 4°C)
 Centrifuge (13000 rpm, 10 min, 4°C)
 Collect SN (= membrane proteins), set aside 50 µl for measurement of protein concentration
 Shock freeze the rest of SN (liquid N2), store at -80°C, discard the pellet
 Proceed with measurement of protein concentration (e.g. BCA assay), SDS-PAGE and WB
Solutions:
Resuspension Buffer stock (2x):
100 mM Tris-Cl, pH 7.4……………..5 ml of 1 M Tris-Cl, pH 7.4
300 mM NaCl………………………..3 ml of 5 M NaCl
10 mM EDTA………………………2 ml of 250 mM EDTA
40 ml MQ
50 ml
Resuspension Buffer:
5 ml…………..Resuspension Buffer stock (2x)
1 tablet………Complete mini protease inhibitor (Roche #11 836 170 001)
5 ml…………..MQ
10 ml
Info Box:
Ripa buffer:
5 ml…………..Resuspension Buffer stock (2x, final:1x)
1 tablet……….Complete mini protease inhibitor (Roche)
1 ml………….10% NP40 (final: 1%, Roche #11332473001)
500 µl………..10% Na-deoxycholate (final: 0.5%)
100 µl………..10% SDS (final: 0.1%)
3.4 ml………...MQ
10 ml
Protein extraction from mammalian cells
RIPA buffer
(RadioImmunoPrecipitation Assay buffer):
RIPA buffer contains the ionic detergent
sodium deoxycholate and NP40 (Nonidet P40, a non-ionic, non-denaturing detergent) as
active constituents.
It is the most commonly used lysis buffer for
membrane disruption in membrane protein
extraction. RIPA buffer gives low background
but can denature kinases. It can also disrupt
protein-protein interaction (and may therefore
be problematic for Immunoprecipitations/pull
down assays).
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