Method for Protein Subfractionation of Cardiovascular Tissues Before DIGE Analysis - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 289

2.2. Extraction Buffers

1. Extraction buffer 1 : 0.5 M NaCl and 10 mM Tris-HCl, pH
7.5 (dilute the 100 mM Tris-HCl stock solution).
2. Extraction buffer 2 : 1% (35 mM) sodium dodecyl sulfate (SDS).
In order to facilitate dissolution of SDS, warm up the buffer
under hot tap water. SDS readily crystallizes at <20°C. Warm
up to redissolve before use.
3. Extraction buffer 3 : 4 M guanidine HCl and 50 mM sodium
acetate. pH should be adjusted to 5.8 using 12 M NaOH.
4. Interstitial collagen extraction buffer : 1 M solution of pure ace-
tic acid. Add 10 g/mL ultrapure pepsin from porcine gastric

mucosa (Sigma-Aldrich, Poole, UK).
5. 4× deglycosylation buffer : 600 mM NaCl, 200 mM Na 2 HPO 4 ,
in ddH 2 O, pH 6.8. Prior to deglycosylation, 0.05 U of the fol-
lowing enzymes are added to the 1× deglycosylation buffer :
Chondroitinase ABC from Proteus vulgaris , which catalyzes
the removal of polysaccharides containing 1

μ

→ 4- β - D -hexo-

saminyl and 1 - L -iduronosyl

linkages to disaccharides containing 4-deoxy-

→ 3- β - D -glucuronosyl or 1 → 3- α

- D -gluc-4-
enuronosyl groups. It acts on chondroitin 4-sulfate, chon-
droitin 6-sulfate, and dermatan sulfate glycosaminoglycan side
chains (Sigma-Aldrich).
Keratanase from Bacteroides fragilis , which cleaves internal

β

1 -galactose linkages in unbranched, repeating poly- N -

acetyl-lactosamine and keratan sulfate (Sigma-Aldrich).

→ 4- β

1. Protein precipitation and sample clean-up: 2DE clean-up kit
(Bio-Rad, Hercules, CA).
2. DIGE lysis buffer: 8 M urea, 30 mM Tris-HCl, pH 8.5, 4%
(w/v) CHAPS, and protease inhibitors. To prepare 100 mL of
DIGE lysis buffer, dissolve 48 g urea in 90 mL of ddH 2 O.
After urea is dissolved, add 3 mL of 1 M Tris and 4 g CHAPS.
Carefully adjust pH to 8.5 with diluted HCl while the buffer is
still cold. Add two predissolved protease inhibitor cocktail tab-
lets (Roche, Burgess Hill, UK) and top up to 100 mL with
ddH 2 O. Mix well, then freeze 1-mL aliquots at −80°C.
3. DIGE 2× buffer: 8 M urea, 2% (v/v) immobilized pH gradient
(IPG) buffer (GE Healthcare), 2% (w/v) DTT, 4% (w/v)
CHAPS. To prepare 100 mL of DIGE 2× buffer, dissolve 48 g
urea in 90 mL of ddH 2 O. Once urea is dissolved, add 2 mL of
IPG buffer (matching the pH of the IPG strips), 2 g DTT, 4 g
CHAPS and top up to 100 mL with ddH 2 O. Mix well, then
freeze 0.5-mL aliquots at −80°C.
4. Rehydration buffer: 8 M urea, 0.5% (w/v) CHAPS, 0.2% (w/v)
DTT, 0.5% (v/v) IPG buffer, trace of bromophenol blue. To
prepare 100 mL of rehydration buffer, dissolve 50 g urea in
water and top up to 100 mL. Once urea is dissolved, add 1 g

2.3. DIGE Analysis