fish to mammalian) and has also been applied in different research
fields (from food technology to nutrition) [7–9] demonstrating its
robustness and reliability.
2 Materials
Prepare all solutions using deionized H 2 O (Milli-Q-purified water
or equivalent) unless indicated otherwise. Prepare and store all
reagents at room temperature unless otherwise specified.
2.1 Preparation
of Protein Extracts
1. For extraction of protein soluble in low ionic strength (LIS),
sodium phosphate buffer (PB): 20 mM sodium phosphate,
0.5 mM magnesium chloride (MgCl 2 ), and 1 mM
ethylenediamine-tetraacetic acid (EDTA), pH 6.0 with protease inhibitor cocktail (see Note 1). Dissolve 286 mg of sodium
phosphate (!99.0%) (246 mg of NaH 2 PO 4 and 39.6 mg of
Na 2 HPO 4 ) in 90 mL (see Note 2), add 4.76 mg of MgCl 2
(anhydrous, !98%), and 41.6 mg of EDTA. Mix and check the
pH. Then, rise the volume up to 100 mL in a volumetric flask.
Protease inhibitor cocktail will be added following commercial
instructions at the moment of performing protein extractions.
Store at 4
C until use.
2. For extraction of protein soluble in high ionic strength (HIS),
Tris Buffer high Saline (TBhS): 10 mM Tris–HCl, 0.6 M
sodium chloride (NaCl), pH 7.2 with protease inhibitor cocktail and EDTA (see Note 1). To prepare 100 mL of buffer,
dissolve 158 mg of Tris–HCl (!99.0%) and 351 mg of NaCl
(!99.0%) in 80 mL H 2 O. Mix and adjust pH (see Note 3).
When pH is adjusted, transfer to a volumetric flask and add
H 2 O up to a volume of 100 mL. Both protease inhibitor
cocktail and EDTA will be added following commercial
instructions at the moment of performing protein extractions.
Store at 4
C until use.
3. Homogenizer for tissue extractions (Ultra-Turrax T-50 or
equivalent) (see Note 4).
4. Refrigerated microcentrifuge.
5. Refrigerated ultracentrifuge.
6. Protein quantification Bradford kit (see Note 5).
7. Spectrophotometer capable of measuring absorbance in the
595 nm region.
2.2 FTSC-Labeling
of Protein-Bound
Carbonyls
1. Sodium phosphate buffer (PB), pH 6 (see Note 6).
2. 250 mM EDTA: dissolve 1.04 g EDTA in 10 mL deionized
water.
230
Lucı ´a Me ´ ndez et al.
fields (from food technology to nutrition) [7–9] demonstrating its
robustness and reliability.
2 Materials
Prepare all solutions using deionized H 2 O (Milli-Q-purified water
or equivalent) unless indicated otherwise. Prepare and store all
reagents at room temperature unless otherwise specified.
2.1 Preparation
of Protein Extracts
1. For extraction of protein soluble in low ionic strength (LIS),
sodium phosphate buffer (PB): 20 mM sodium phosphate,
0.5 mM magnesium chloride (MgCl 2 ), and 1 mM
ethylenediamine-tetraacetic acid (EDTA), pH 6.0 with protease inhibitor cocktail (see Note 1). Dissolve 286 mg of sodium
phosphate (!99.0%) (246 mg of NaH 2 PO 4 and 39.6 mg of
Na 2 HPO 4 ) in 90 mL (see Note 2), add 4.76 mg of MgCl 2
(anhydrous, !98%), and 41.6 mg of EDTA. Mix and check the
pH. Then, rise the volume up to 100 mL in a volumetric flask.
Protease inhibitor cocktail will be added following commercial
instructions at the moment of performing protein extractions.
Store at 4
C until use.
2. For extraction of protein soluble in high ionic strength (HIS),
Tris Buffer high Saline (TBhS): 10 mM Tris–HCl, 0.6 M
sodium chloride (NaCl), pH 7.2 with protease inhibitor cocktail and EDTA (see Note 1). To prepare 100 mL of buffer,
dissolve 158 mg of Tris–HCl (!99.0%) and 351 mg of NaCl
(!99.0%) in 80 mL H 2 O. Mix and adjust pH (see Note 3).
When pH is adjusted, transfer to a volumetric flask and add
H 2 O up to a volume of 100 mL. Both protease inhibitor
cocktail and EDTA will be added following commercial
instructions at the moment of performing protein extractions.
Store at 4
C until use.
3. Homogenizer for tissue extractions (Ultra-Turrax T-50 or
equivalent) (see Note 4).
4. Refrigerated microcentrifuge.
5. Refrigerated ultracentrifuge.
6. Protein quantification Bradford kit (see Note 5).
7. Spectrophotometer capable of measuring absorbance in the
595 nm region.
2.2 FTSC-Labeling
of Protein-Bound
Carbonyls
1. Sodium phosphate buffer (PB), pH 6 (see Note 6).
2. 250 mM EDTA: dissolve 1.04 g EDTA in 10 mL deionized
water.
230
Lucı ´a Me ´ ndez et al.