May 15, 2023

Public workspaceCompartmental Protein Extraction to Achieve Enrichment of Extracellular Matrix (ECM) Proteins

DOI
dx.doi.org/10.17504/protocols.io.kxygx94b4g8j/v1
  • 1University of Illinois Chicago
Alexandra Naba
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DOI: dx.doi.org/10.17504/protocols.io.kxygx94b4g8j/v1
Protocol Citation: James Considine, Ikram Isa, Alexandra Naba 2023. Compartmental Protein Extraction to Achieve Enrichment of Extracellular Matrix (ECM) Proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.kxygx94b4g8j/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working!
Created: March 14, 2023
Last Modified: May 22, 2023
Protocol Integer ID: 78808
Keywords: Protein Extraction, Extracellular Matrix, Decellularization
Abstract
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.


https://www.youtube.com/embed/JrPH9FDY05c

Materials
I. Materials & Reagents

1. ReagentTissue homogenizer: Bullet blender + beadsNext AdvanceCatalog #BB24-AU OR ReagentTissue homogenizer: Bead Rupter Elite + BeadsOmni InternationalCatalog #19-042E

2. ReagentSubcellular Protein Fractionation Kit for TissuesThermo ScientificCatalog #87790

3. ReagentBupH Phosphate Buffered Saline PacksThermo ScientificCatalog #28372



Note
  • The protocol uses a series of incubation in buffers of different pH and containing different amounts of salts and detergents to sequentially extract intracellular proteins and enrich for insoluble ECM proteins.

  • The volumes of reagents are from Pierce Compartmental given below are for 100mg of tissues or tumors (see Table 1) and need to be adjusted appropriately.

  • A cocktail of protease inhibitors (PI, Cat#1862209) is provided as a 100X solution and needs to be added to each buffer. Protease inhibitor: A cocktail of various protease inhibitors is included with the kit, but if this is not used it is advisable to include a variety of inhibitors against cysteine, serine and threonine peptidases, serine esterases, divalent cation-dependent metalloproteinases etc

  • We do not recommend conducting the procedure on fixed tissues as fixation (i.e., chemical crosslinking) interferes with decellularization and can also significantly compromise subsequent mass spectrometry analysis.

  • For the entire procedure, we recommend the use of low-retention tubes and pipette tips to maximize protein recovery.













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.
Tissue Homogenization
Tissue Homogenization
10m
10m
Homogenize Amount100 mg of tissue in Amount1 mL 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.

Expected result
Post Homogenization Example



Sequential extraction of intracellular soluble proteins
Sequential extraction of intracellular soluble proteins
2h 10m
2h 10m

2h 10m
Extraction of cytosolic proteins:
  1. Centrifuge the homogenate at Centrifigation5000 x g for Duration00:10:00 at Temperature4 °C .
  2. Collect the supernatant in a clean tube: this fraction is enriched for cytosolic (C) proteins. Flash freeze this fraction and store at Temperature-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.

Expected result
Post extraction of cytosolic proteins pellet


10m
Extraction of membrane proteins:
  1. Resuspend the pellet in Amount650 µL of Buffer MEB containing protease inhibitors.
  2. Incubate the sample on a tube rotator for Duration00:10:00 at Temperature4 °C
  3. Centrifuge the sample at Centrifigation5000 x g for Duration00:10:00 atTemperature4 °C
  4. Collect the supernatant in a clean tube: this fraction is enriched for membrane (M) proteins. Flash-freeze this fraction and store at Temperature-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.


Expected result
Post extraction of membrane proteins pellet





20m
Extraction of nuclear soluble proteins:
  1. Resuspend the pellet in Amount225 µL of Buffer NEB containing protease inhibitors.
  2. Incubate the sample on a tube rotator for Duration00:30:00 at Temperature4 °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.

  1. Centrifuge the sample at Centrifigation5.000 x g for Duration00:10:00 atTemperature4 °C
  2. Collect the supernatant in a clean tube: this fraction is enriched for nuclear soluble (Nsol) proteins. Flash-freeze this fraction and store at Temperature-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.


Expected result
Post extraction of nuclear soluble proteins pellet




40m
Extraction of nuclear chromatin-bound proteins:
  1. Resuspend the pellet in Amount170 µL of Buffer NEB containing protease inhibitors, CaCl2, and micrococcal nuclease.
  2. Incubate the sample on a tube rotator forDuration00:30:00 atTemperatureRoom temperature
  3. Centrifuge the sample at Centrifigation16000 x g for Duration00:10:00 at Temperature4 °C
  4. Collect the supernatant in a clean tube: this fraction is enriched for nuclear chromatin-bound (Ncb) proteins. Flash-freeze this fraction and store at Temperature-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.

Expected result
Post extraction of nuclear insoluble proteins pellet



40m


Extraction of cytoskeletal proteins:
  1. Resuspend the pellet in Amount125 µL of Buffer PEB containing protease inhibitors.
  2. Incubate the sample on a tube rotator forDuration00:20:00 at TemperatureRoom 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 Centrifigation16000 x g for Duration00:10:00 atTemperatureRoom temperature .
4. Collect the supernatant in a clean tube: this fraction is enriched for cytoskeletal (Cs) proteins

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.


Expected result
Post extraction of cytoskeletal proteins pellet





30m
Washing the remaining pellet enriched for ECM proteins
Washing the remaining pellet enriched for ECM proteins
10m
10m
All traces of detergents need to bTemperature4 °C 4 °C removed by extensive washes prior to digestion of the proteins into peptides for mass spectrometric analysis. Perform washes as follows:
  1. Resuspend the pellet in Amount600 µL of PBS containing protease inhibitors.
  2. Centrifuge the sample at Centrifigation16000 x g for Duration00:03:00 atTemperature4 °C
  3. Discard the supernatant
  4. 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.

Expected result
Post washing pellet




9m
Sample storage
Sample storage
5m
5m
The ECM-enriched pellet can be flash-frozen and kept atTemperature-80 °C . The ECM-enriched protein pellet can be subsequently digested into peptides for proteomic analysis:
Protocol
In-solution Digestion of ECM-Enriched Proteins Samples for Mass Spectrometry Analysis
NAME
In-solution Digestion of ECM-Enriched Proteins Samples for Mass Spectrometry Analysis
CREATED BY
Alexandra Naba




5m
Protocol references