TITLE: Compartmental Protein Extraction to Achieve Enrichment of Extracellular Matrix (ECM) Proteins
AUTHORS: James Considine, Ikram Isa, Alexandra Naba

[DESCRIPTION]
This protocol provides instructions on how to extract cellular components of tissues and enrich for ECM proteins to be later analyzed via immunoblotting or mass spectrometry. 


 


[BEFORE_START]

 
Before starting, prepare the reagents and add protease inhibitors provided with the Subcellular Protein Fractionation Kit for Tissues to the desired volume of each buffer. 

All buffers and samples should be kept on ice for the duration of the experiment except the Buffer PEB that needs to be kept at room temperature to prevent SDS precipitation.

[STEPS]
SECTION: Tissue Homogenization
1. Homogenize 100 mg  of tissue in 1 mL  of Buffer CEB containing protease inhibitors using a tissue homogenizer until the tissue is completely disrupted and a homogenous suspension is obtained. 

 

 


SECTION: Sequential extraction of intracellular soluble proteins
2. 
SECTION: Sequential extraction of intracellular soluble proteins
2.1. Extraction of cytosolic proteins:
Centrifuge the homogenate at 5000 x g for 10 min at 4 °C. 
Collect the supernatant in a clean tube: this fraction is enriched for cytosolic (C) proteins. Flash freeze this fraction and store at -80 °C.



 

 

SECTION: Sequential extraction of intracellular soluble proteins
2.2. Extraction of membrane proteins:
Resuspend the pellet in 650 µLof Buffer MEB containing protease inhibitors.
Incubate the sample on a tube rotator for 10 min at 4 °C
Centrifuge the sample at 5000 x g for 10 min  at4 °C
Collect the supernatant in a clean tube: this fraction is enriched for membrane (M) proteins. Flash-freeze this fraction and store at -80 °C



 


 




SECTION: Sequential extraction of intracellular soluble proteins
2.3.  Extraction of nuclear soluble proteins:
 Resuspend the pellet in 225 µL of Buffer NEB containing protease inhibitors.
 Incubate the sample on a tube rotator for 30 minat 4 °C

 

Centrifuge the sample at 5.000 x g for 10 min at4 °C
Collect the supernatant in a clean tube: this fraction is enriched for nuclear soluble (Nsol) proteins. Flash-freeze this fraction and store at -80 °C



 


 



SECTION: Sequential extraction of intracellular soluble proteins
2.4. Extraction of nuclear chromatin-bound proteins:
Resuspend the pellet in 170 µL of Buffer NEB containing protease inhibitors, CaCl2, and micrococcal nuclease.
Incubate the sample on a tube rotator for30 min at4 Room temperature
Centrifuge the sample at 16000 x g for 10 min at 4 °C
Collect the supernatant in a clean tube: this fraction is enriched for nuclear chromatin-bound (Ncb) proteins. Flash-freeze this fraction and store at -80 °C






 

 


SECTION: Sequential extraction of intracellular soluble proteins
2.5. 

 Extraction of cytoskeletal proteins:
 Resuspend the pellet in 125 µLof Buffer PEB containing protease inhibitors. 
Incubate the sample on a tube rotator for20 min at Room temperature.

 
3. Centrifuge the sample at 16000 x g for 10 min atRoom temperature. 
4. Collect the supernatant in a clean tube: this fraction is enriched for cytoskeletal (Cs) proteins

 

 




SECTION: Washing the remaining pellet enriched for ECM proteins
3. All traces of detergents need to b4 °C removed by extensive washes prior to digestion of the proteins into peptides for mass spectrometric analysis. Perform washes as follows:
Resuspend the pellet in 600 µL of PBS containing protease inhibitors. 
Centrifuge the sample at 16000 x gfor 3 min at4 °C
Discard the supernatant
Repeat this step two times
 

 



SECTION: Sample storage
4. The ECM-enriched pellet can be flash-frozen and kept at-80 °C. The ECM-enriched protein pellet can be subsequently digested into peptides for proteomic analysis: