Sodium chloride
(NaCl), trifluoroacetic acid (TFA), lactic acid, calcium chloride
(CaCl2), hydrochloric acid (HCl, 36%), acetonitrile (ACN),
urea (NH2CONH2), aminodiacetic acid (HN(CH2COOH)2), ammonia solution (NH4OH, 25%),
methyl cellulose (MC, viscosity: 1500 cP), and sodium hydroxide (NaOH)
were all purchased from Sigma-Aldrich (USA). Acetic acid and phosphoric
acid (85 wt %) were brought from Merck (USA); sinapic acid (SA) was
provided by Supelco (USA). The amphoteric electrolytes: Pharmalyte
3–10 carrier ampholytes were purchased from Cytiva (USA). The
pI markers (pH = 5.500 and 3.210) were purchased from AB Sciex (USA)
and AES (Canada). The tris(hydroxymethyl)aminethane (THAM) solution
of 1 M was purchased from Thermo Fisher (USA). Lactoferrin (L9507),
α-lactalbumin (L5385), β-lactoglobulin (L3908), bovine
serum albumin (BSA), α-casein (C6780), β-casein (C6905),
κ-casein (C0406), and lysozyme (L6876) were all purchased from
Sigma-Aldrich (USA). Casein glycomacropeptide (CGMP) was provided
by Agropur (BiPRO GMP 9000). Casein phosphopeptides (CPP) were provided
by Ingredia Nutritional (OSTEUM CPP). All of the reagents were directly
used for experiments without further purification.
The cIEF
gel was obtained by mixing 15 mL of ultrapure water and 0.4 g of MC
powder, followed by stirring for 5 min at 80 °C. After the mixture
was removed from the heater, an ice–water mixture was added
to the solution to 40 mL. Then, the solution was stirred every 30
min until cooling to −20 °C. Then, the obtained mixture
was stored at 4–8 °C overnight. Furthermore, 3 M urea
gel solution was prepared by dissolving 1.8 g of urea and 6 mL of
cIEF gel with ultrapure water added, meeting a final volume of 10
mL. After mixing, the as-prepared solution was stored at 4 °C.
(NaCl), trifluoroacetic acid (TFA), lactic acid, calcium chloride
(CaCl2), hydrochloric acid (HCl, 36%), acetonitrile (ACN),
urea (NH2CONH2), aminodiacetic acid (HN(CH2COOH)2), ammonia solution (NH4OH, 25%),
methyl cellulose (MC, viscosity: 1500 cP), and sodium hydroxide (NaOH)
were all purchased from Sigma-Aldrich (USA). Acetic acid and phosphoric
acid (85 wt %) were brought from Merck (USA); sinapic acid (SA) was
provided by Supelco (USA). The amphoteric electrolytes: Pharmalyte
3–10 carrier ampholytes were purchased from Cytiva (USA). The
pI markers (pH = 5.500 and 3.210) were purchased from AB Sciex (USA)
and AES (Canada). The tris(hydroxymethyl)aminethane (THAM) solution
of 1 M was purchased from Thermo Fisher (USA). Lactoferrin (L9507),
α-lactalbumin (L5385), β-lactoglobulin (L3908), bovine
serum albumin (BSA), α-casein (C6780), β-casein (C6905),
κ-casein (C0406), and lysozyme (L6876) were all purchased from
Sigma-Aldrich (USA). Casein glycomacropeptide (CGMP) was provided
by Agropur (BiPRO GMP 9000). Casein phosphopeptides (CPP) were provided
by Ingredia Nutritional (OSTEUM CPP). All of the reagents were directly
used for experiments without further purification.
The cIEF
gel was obtained by mixing 15 mL of ultrapure water and 0.4 g of MC
powder, followed by stirring for 5 min at 80 °C. After the mixture
was removed from the heater, an ice–water mixture was added
to the solution to 40 mL. Then, the solution was stirred every 30
min until cooling to −20 °C. Then, the obtained mixture
was stored at 4–8 °C overnight. Furthermore, 3 M urea
gel solution was prepared by dissolving 1.8 g of urea and 6 mL of
cIEF gel with ultrapure water added, meeting a final volume of 10
mL. After mixing, the as-prepared solution was stored at 4 °C.
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