From Fey et al. (1984) JCB 99: 203s-208s.

A 3-Step process for removal of non-IF material in cells that leaves only 5% of original cell protein, almost all of which is IFs (both cytoskeletal and lamins)

Summary
1.    Extract soluble fraction with harsh detergent (0.5% Triton X-100)
2.    Extract salt-labile fraction with 0.25 M (NH4)2SO4
3.    Extract nuclear proteins and nucleic acids with DNase and RNase

Buffer 1 – Soluble Fraction
100 mM NaCl
300 mM sucrose
10 mM PIPES (pH 6.8)
3 mM MgCl2
0.5% Triton X-100
1.1    mM PMFS
protease inhibitor cocktail (1 ml to 100 mL)

Buffer 2 – Salt Labile Fraction (non-IF CSK mostly)
250 mM (NH4)2SO4
300 mM sucrose
10 mM PIPES (pH 6.8)
3 mM MgCl2
0.5% Triton X-100
1.1    mM PMFS
protease inhibitor cocktail (1 ml to 100 mL)

Buffer 3 – Nuclear Fraction (nucleic acids and proteins)
Same as Buffer 1, PLUS:
50 mM NaCl (not 100 mM)
100 μg/ml bovine pancreatic DNase
100 μg/ml bovine pancreatic Rnase
protease inhibitor cocktail (1 ml to 100 mL)

PROTOCOL:

• Rinse cells with PBS + 0.5 mM MgCl2 + 1 mM CaCl2
• Cover with Buffer 1 for 10 minutes at 0 degrees on rocker
• Replace with Buffer 2 for 10 minutes at 0 degrees on rocker
• Replace Buffer 2 with Buffer 3, incubate for 20 min. at RT.
• Add (NH4)2SO4 to a final concentration of 0.25 M, incubate for 5 more min.
• Rinse with PBS +Ca+Mg
• Fix for 30 min. at 0 degrees with 3% paraformaldehyde for immunofluorescence, or 3% paraformaldehyde + 3% glutaraldehyde for EM or AFM.

STOCK SOLUTIONS:
1.5 M NaCl    dissolve 21.92 g NaCl in 250 mL dH2O
0.2 M MgCl2    dissolve 10.16 g MgCl2 in 250 mL dH2O
0.2 M CaCl2    dissolve 7.35 g MgCl2 in 250 mL dH2O
0.2 M PIPES    dissolve 15.12 g PIPES in 250 mL dH2O, pH 6.8
4.0 M (NH4)2SO4    dissolve 132.1 g (NH4)2SO4 in 250 mL dH2O
0.153 M PMSF    dissolve 0.04 g in 1.5 mL of ethanol

WORKING SOLUTIONS:
Buffer 1 – for 100 mL
100 mM NaCl    Add 6.67 mL of 1.5 M stock
10 mM PIPES (pH 6.8)    Add 5.0 mL of 0.2 M stock
1 mM CaCl2    Add 0.5 mL of 0.2 M stock
3 mM MgCl2    Add 1.5 mL of 0.2 M stock
bring up to about 70 mL in a 100 mL beaker    
0.5% Triton X-100    Add 0.5 mL Triton X-100
300 mM sucrose    Add 10.27 g sucrose
1.1 mM PMFS    Add 0.75 mL of 0.14 M PMFS stock
protease inhibitor cocktail    Add 1.0 mL
bring up to final volume of 100 mL with dH2O and adjust pH to 6.8

Buffer 2 – for 100 mL
250 mM (NH4)2SO4        Add 6.25 mL of 4 M stock
3 mM MgCl2            Add 1.5 mL of 0.2 M stock
10 mM PIPES (pH 6.8)     Add 5.0 mL of 0.2 M stock
bring up to about 70 mL in a 100 mL beaker
0.5% Triton X-100        Add 0.5 mL Triton X-100
300 mM sucrose        Add 10.27 g sucrose 
1.1 mM PMFS        Add 0.75 mL of 0.14 M PMFS stock
protease inhibitor cocktail    Add 1.0 mL
bring up to final volume of 100 mL with dH2O and adjust pH to 6.8

Buffer 3 – for 100 mL 
100 mM NaCl        Add 3.33 mL of 1.5 M stock
10 mM PIPES (pH 6.8)    Add 5.0 mL of 0.2 M stock
1 mM CaCl2            Add 0.5 mL of 0.2 M stock
3 mM MgCl2            Add 1.5 mL of 0.2 M stock
bring up to about 70 mL in a 100 mL beaker
0.5% Triton X-100        Add 0.5 mL Triton X-100
300 mM sucrose        Add 10.27 g sucrose
1.1 mM PMFS        Add 0.75 mL of 0.14 M PMFS stock
protease inhibitor cocktail    Add 1.0 mL
add 0.010 g bovine pancreatic DNase (depends on stock concentration)
add 0.010 g bovine pancreatic RNase (depends on stock concentration)
bring up to final volume of 100 mL with dH2O and adjust pH to 6.8

*protease inhibition is critical due to presence of Ca++ in buffer, which activates lots of proteases.