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Protocol - Cytoplasmic Protein extraction

S100 cytoplasmic extract

Category: Molecular Biology

Creation date: Aug 07, 2014

Last revision: May 26, 2015

Author(s): Laura Pisapia, Pasquale Barba, Giovanna Del Pozzo

Contact

Name: Giovanna Del Pozzo

Address: IGB- CNR

Phone: 0816132309

Email: giovanna.delpozzo@igb.cnr.it

Steps

Description	Temperature	Time
Use at least 30 million cultured cells (for this protocol the recommended number is between 30 and 60 million)	room temperature
Harvest cells and wash with 5-10 ml cold pPBS and centrifuge at 200 g	4°C
Wash cells with 5-10 ml of cold hypotonic buffer and centrifuge at 200 g	4°C	5 min
Suspend the cells in 3-4 ml of hypotonic buffer and incubate on ice, stirring rarely	4°C	5 min
Transfer the cells to a glass Dounce homogenizer. Homogenize with 10-20 up-and-down strokes using a loose-fitting pestle	4°C	5-10 min
After homogenization, add 20 ul of sample to 180 ul of diluted Tryplan blu and check by counting unstained (viable) and stained (dead) cells. If the dead cells are lower than the 70%, homogenise again with five up-and-down strokes and repeat trypan blue count . Repeat until lysis is 80- 90 %	4°C	10 min
Total volume of extracts were aliquoted in eppendorf tubes and centrifuged at 3300g to pellet nuclei and recovered cytoplasmic supernatant.	4°C	15 min
The supernatant was mixed thoroughly with 0.11 volumes of 10x cytoplasmic extraction buffer and was centrifuged at 100 000g in a Beckman ultracentrifuge using Beckman Type 55SW rotor(32400 rpm)	4°C	1 hr
The recovered supernatant was dialysed against 50 volumes of dialysis buffer twice, for 1 hr and over night	4°C	1 hr and over night
After dialysis the protein extract concentration will be measured by BREADFORD assay and protein will be aliquoted and stored at -80°C	room temperature	30 min

Quality validation: Yes

The protocol has been used to produce data published in peer-reviewed journals.

Citations
Pisapia L, Cicatiello V, Barba P, Malanga D, Maffei A, Hamilton RS, Del Pozzo G. Co-regulated expression of alpha and beta mRNAs encoding HLA-DR surface heterodimers is mediated by the MHCII RNA operon. Nucleic Acids Res. 2013;41:3772-86.
Corso C, Pisapia L, Citro A, Cicatiello V, Barba P, Cigliano L, et al. EBP1 and DRBP76/NF90 binding proteins are included in the major histocompatibility complex class II RNA operon. Nucleic Acids Res 2011;39:7263-7275.

Related Protocols:

Reagents	F-concentration	Quantity	I-concentration
HEPES, pH 7.9	0,3M
KCl	1,4 M
MgCl2	0,03M
PI (protease inhibitors cocktail ROCHE)	1X

Add PI before use

Reagents	F-concentration	Quantity	I-concentration
HEPES, pH 7.9	10 mM
MgCl2	1.5 mM
KCl	10mM
PMSF	0,2 mM
PI (protease inhibitors cocktail ROCHE)	1x