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.
#尊敬的用户，由于网络监管政策的限制，部分内容暂时无法在本网站直接浏览。我们已经为您准备了相关原始数据和链接，感谢您的理解与支持。
<iframe width="560" height="315" src="https://www.youtube.com/embed/JrPH9FDY05c" title="Enrichment Of Extracellular Matrix Proteins From Tissues & Digestion: Peptides l Protocol Preview" frameborder="0" allow="accelerometer; autoplay; clipboard-write; encrypted-media; gyroscope; picture-in-picture; web-share" allowfullscreen></iframe>
Before start
Note
The procedure can be conducted on fresh or flash-frozen tissues. We recommend perfusing highly vascularized tissues with PBS at the time of tissue collection to eliminate red blood cells and plasma proteins.
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
Tissue Homogenization
1.
Homogenize 100mg of tissue in 1mL of Buffer CEB containing protease inhibitors using a tissue homogenizer until the tissue is completely disrupted and a homogenous suspension is obtained.
Note
Save a small 50 uL aliquot of the homogenate and add 50 uL of 3X Laemmli buffer with 100 mM DTT to monitor the quality of the protein extraction by western blotting. Flash freeze and store at -80C.
Citation
Post Homogenization Example
Sequential extraction of intracellular soluble proteins
2.
2.1.
Extraction of cytosolic proteins:
Centrifuge the homogenate at 5000x g,0h 0m 0s for 0h 10m 0s 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.
Note
Aliquot 50 uL of the supernatant and add 25 uL of 3X Laemmli Buffer with 100 mM DTT to monitor the quality of the extraction by western blotting. Flash freeze and store at -80 C.
Citation
Post extraction of cytosolic proteins pellet
2.2.
Extraction of membrane proteins:
Resuspend the pellet in 650µLof Buffer MEB containing protease inhibitors.
Incubate the sample on a tube rotator for 0h 10m 0s at 4°C
Centrifuge the sample at 5000x g,0h 0m 0s for 0h 10m 0s 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
Note
Aliquot 50 uL of the supernatant and add 25 uL of 3X Laemmli Buffer with 100 mM DTT to monitor the quality of the extraction by western blotting. Flash freeze and store at -80 C.
Citation
Post extraction of membrane proteins pellet
2.3.
Extraction of nuclear soluble proteins:
Resuspend the pellet in 225µL of Buffer NEB containing protease inhibitors.
Incubate the sample on a tube rotator for 0h 30m 0sat 4°C
Note
Buffer NEB containing protease inhibitors, CaCl2, and micrococcal nuclease should be taken off the ice at this point in preparation for step 2.4.
Centrifuge the sample at 5.000x g,0h 0m 0s for 0h 10m 0s 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
Note
Aliquot 50 uL of the supernatant and add 25 uL of 3X Laemmli Buffer with 100 mM DTT to monitor the quality of the extraction by western blotting. Flash freeze and store at -80 C.
Citation
Post extraction of nuclear soluble proteins pellet
2.4.
Extraction of nuclear chromatin-bound proteins:
Resuspend the pellet in 170µL of Buffer NEB containing protease inhibitors, CaCl2, and micrococcal nuclease.
Incubate the sample on a tube rotator for0h 30m 0s at4Room temperature
Centrifuge the sample at 16000x g,0h 0m 0s for 0h 10m 0s 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
Note
Aliquot 50 uL of the supernatant and add 25 uL of 3X Laemmli Buffer with 100 mM DTT to monitor the quality of the extraction by western blotting. Flash freeze and store at -80 C.
Citation
Post extraction of nuclear insoluble proteins pellet
2.5.
Extraction of cytoskeletal proteins:
Resuspend the pellet in 125µLof Buffer PEB containing protease inhibitors.
Incubate the sample on a tube rotator for0h 20m 0s at Room temperature.
Note
Note that the pellet may not fully resuspend. We suggest disrupting the pellet by pipetting up and down until observing disruption of the pellet. To facilitate disruption, we suggest using cut tips.
3. Centrifuge the sample at 16000x g,0h 0m 0s for 0h 10m 0s atRoom temperature.
Collect the supernatant in a clean tube: this fraction is enriched for cytoskeletal (Cs) proteins
Note
Note at this point a further marked decrease in the size of the pellet (see video available at: Note at this point a further marked decrease in the size of the pellet (see video available at: Enrichment of ECM Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis))Aliquot 50 uL of the supernatant and add 25 uL of 3X Laemmli Buffer with 100 mM DTT to monitor the quality of the extraction by western blotting. Flash freeze and store at -80 C.
Citation
Post extraction of cytoskeletal proteins pellet
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 600µL of PBS containing protease inhibitors.
Centrifuge the sample at 16000x g,0h 0m 0sfor 0h 3m 0s at4°C
Discard the supernatant
Repeat this step two times
Note
On the final wash, aliquot 150-200 uL of the 600 uL resuspended ECM-enriched pellet to monitor the quality of the extraction by western blotting. Centrifuge aliquot at 16,000 x g for 3 minutes, and resuspend in 25-50 uL of 3x Laemmli buffer with 100 mM of DTT. Flash freeze and store at -80 C.Note that the size of the pellet will depend on the amount of insoluble (ECM) proteins in the starting material and the efficiency of the decellularization.
Citation
Post washing pellet
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:
