1. Preparation of Cells
DDF can be used to separate cells grown in liquid suspension or on a flat surface. After DDF, all extracts should be stored frozen at –70°C. A portion of DDF buffers should be saved at –70°C for normalization of samples and as a control material for enzyme and protein analysis. To avoid any impact from culture media, cells should be washed twice in a non-detergent buffer, ice-cold saline or PBS before DDF.
1.1. Suspension-Cultured Cells
1) For cells grown in liquid suspension, the volume of DDF solutions is determined by wet weight or cell count. To determine wet weight, a sample of liquid culture should be transferred to a pre-weighed plastic tube and centrifuged briefly.
2) After removing the culture media, the wet weight of the cell pellet is determined. Digitonin/EDTA extraction buffer (5 volumes/g wet weight) can be added directly to the cell pellet.
1.2. Monolayer-Cultured Cells
1) The volume of DDF buffers for cells grown on a flat surface is determined by surface area or cell count. For a typical T25 culture flask (about 5 × 106 cells), 1 mL of digitonin/EDTA buffer is initially used.
2) After removing the culture medium, DDF can be performed in the culture flask.
2. Detergent Fractionation
2.1. Digitonin Extraction (Cytosolic Fraction)
1) Add ice-cold digitonin extraction buffer (5 volumes/g wet weight) to washed cell pellets (gently resuspend by swirling) or monolayers (1 mL/T25 flask).
2) Incubate the cells on ice with gentle agitation (platform mixer) until 95–100% of cells are permeabilized (5–10 min), as determined by trypan blue exclusion.
3) For cells grown in liquid suspension, centrifuge the extraction mixture (480g) and remove the supernatant.
4) For cell monolayers, tilt the culture flask and remove the extract (cytosolic proteins) with a pipette. Record the extract volume, aliquot, and store at –70°C.
2.2. Triton X-100 Extraction (Membrane/Organelle Fraction)
1) Carefully resuspend digitonin-insoluble pellets in ice-cold Triton X-100 extraction buffer in a volume equivalent to that used for digitonin extraction (5 vols relative to starting wet weight) to obtain a homogeneous suspension.
2) For monolayer cultures, add 1 mL Triton extraction buffer per T25 flask equivalent (approx 5 × 106 cells).
3) Incubate on ice with gentle agitation (platform mixer) for 30 min.
4) Remove Triton extract (membrane and organellar proteins) by centrifuging suspensions (10 min, 5000g) or tilting and decanting monolayers.
5) Measure volume of the extract, aliquot, and store at –70°C.
2.3. Tween/DOC Extraction (Nuclear Fraction)
1) Resuspend the Triton-insoluble pellets from suspension cultures in Tween/DOC extraction buffer at one-half the volume used for Triton extraction; resuspend using a Teflon smooth-walled glass homogenizer (five strokes, medium speed).
2) Remove Tween/DOC extract (nuclear proteins) by pelleting detergent-resistant residue (6780g).
3) Extract cell monolayers with 0.5–1 mL Tween/DOC buffer per T25 flask equivalent.
4) Record the volume, aliquot, and store at –70°C.
2.4. Detergent-Resistant Residue (Cytoskeletal/Nuclear Matrix Fraction)
1) The detergent-resistant pellet is washed in ice-cold PBS (pH 7.4, 1.2 mM PMSF) by resuspension (Teflon/glass homogenizer) and centrifugation (12,000g) to mechanically shear DNA.
2) Pellets from suspension cultures are washed once with –20°C 90% acetone, lyophilized, and weights determined in tared Eppendorf centrifuge tubes. Samples are stored at –70°C.
3) For monolayers, the detergent-resistant residue is rinsed with PBS and directly suspended in nondenaturing cytoskeleton (CSK) solubilization buffer without β-mercaptoethanol by titration, and stored at -70°C.
3. Protein Determination
1) Thaw aliquots of detergent buffers and detergent extracts on ice.
2) Dilute an aliquot of digitonin and Triton X-100 extracts with 4 vols of ultrapure water (extracts obtained from monolayer culture may require less dilution). The Tween/DOC extract is used without dilution.
3) Solubilize lyophilized CSK pellets in CSK solubilization buffer minus β-mercaptoethanol (10 mg dry weight/mL). CSK preparations from monolayers are diluted as necessary.
4) Assay 20–50 μL of diluted or undiluted sample from each fraction, in duplicate, using the Folin-phenol method of Peterson.
5) Use oven-dried bovine serum albumin prepared in each detergent buffer to generate standard curves.
4. Two-Dimensional Gel Electrophoretic Analysis
DDF samples obtained from various cell types can be used for two-dimensional polyacrylamide gel electrophoresis (PAGE) under both isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE). Prior to comparison, samples are normalized to contain equal protein in equal volumes.
4.1. Sample Preparation
1) Keep fresh or defrosted DDF samples on ice.
2) Bring samples obtained from digitonin, Triton X-100, and Tween-40/DOC extracts to 9.5 M urea by adding solid urea.
3) Warm 100 μL sample to room temperature after adding 85 mg urea and 15 μL 10X O'Farrell lysis buffer.
4) Solubilize the dried CSK extract directly in 1X O'Farrell lysis buffer.
5) For CSK extracts in nonreducing SDS buffer, bring to 9.5 M urea by adding solid urea and add 10X lysis buffer.
4.2. Sample Normalization
Samples for comparison are normalized with respect to protein concentration prior to addition of lysis buffers or urea by volume normalization using the appropriate detergent extraction buffer.
4.3. 2-D Electrophoresis
DDF samples are subjected to 2-D gel electrophoresis by established methods. IEF gels contain a total of 3.5% ampholines (2% pH 5.0–8.0, 1% pH 3.0–10.0, 0.5% pH 2.0–5.0), and samples are electrophoresed for a total of 9800 V-h with hyperfocusing at 800 V for the final hour. NEPHGE gels contain 2% pH 3.0–10.0 ampholines, and samples are electrophoresed for 2400 V-h.
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Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.