10. Then, add 87 μL of 100 mM Tris–HCl, pH 5.5.
11. NP-serum solution is incubated at 25
C with shaking
(300 rpm) in a thermostatic bath for 30 min.
12. The pellet is harvested by centrifugation at 15,000 Â g for
30 min.
13. The pellet is washed three times with 50 μL of 100 mM Tris–
HCl, pH 5.5.
14. The pellet is harvested again by centrifugation at 15,000 Â g
for 30 min to remove unbound proteins.
Nanoparticles in Urine
Samples
Au Nanoparticles
Pretreatment of Fresh Urine Sample
1. 20 mL of urine samples is centrifuged at 5000 Â g for 30 min.
2. 3 mL of centrifuged urine is diluted to 30 mL with ultrapure
water, and then urine is aliquoted in five subsamples and stored
at À60
C.
Synthesis of Citrate-Capped Gold Nanoparticles (AuNPs)
Gold nanoparticles, AuNPs, are synthesized by the citrate reduction method in aqueous solution [111].
Protein Reduction and Alkylation
3. The urine is 1/30 diluted with ultrapure water; then, the
sample is adjusted to pH 8.8 with 2.5 M Tris.
4. 600 μL of the sample is reduced with DTT (final concentration
10 mM) and incubated at 37
C for 1 h.
5. Proteins are alkylated with IAA (final concentration 50 mM) at
room temperature for 45 min.
6. After protein reduction and alkylation, 1 mL of AuNPs is added
to the sample and pH is adjusted to 4.0 with 1 M of citric acid.
7. The samples are incubated for 2 h at 37
C in a
thermostatic bath.
8. AuNP–protein complexes are harvested by centrifugation at
8600 Â g for 40 min.
9. The pellet is washed twice with 1 mL of citrate/citric acid
buffer (pH 4.0) to remove unbound protein.
3.3 SDS Gels
1. Protein pellets are reconstituted in 24 μL of Milli-Q water and
are mixed with 6 μL of SDS-PAGE loading buffer in a final
volume of 30 μL.
2. The samples are denatured by heating at 100
C for 5 min and
loaded into a 10% acrylamide/bis-acrylamide, stacking
gel/12.5% acrylamide/bis-acrylamide running gel, of 1 mm
126
Maria del Pilar Chantada-Va ´ zquez et al.
11. NP-serum solution is incubated at 25
C with shaking
(300 rpm) in a thermostatic bath for 30 min.
12. The pellet is harvested by centrifugation at 15,000 Â g for
30 min.
13. The pellet is washed three times with 50 μL of 100 mM Tris–
HCl, pH 5.5.
14. The pellet is harvested again by centrifugation at 15,000 Â g
for 30 min to remove unbound proteins.
Nanoparticles in Urine
Samples
Au Nanoparticles
Pretreatment of Fresh Urine Sample
1. 20 mL of urine samples is centrifuged at 5000 Â g for 30 min.
2. 3 mL of centrifuged urine is diluted to 30 mL with ultrapure
water, and then urine is aliquoted in five subsamples and stored
at À60
C.
Synthesis of Citrate-Capped Gold Nanoparticles (AuNPs)
Gold nanoparticles, AuNPs, are synthesized by the citrate reduction method in aqueous solution [111].
Protein Reduction and Alkylation
3. The urine is 1/30 diluted with ultrapure water; then, the
sample is adjusted to pH 8.8 with 2.5 M Tris.
4. 600 μL of the sample is reduced with DTT (final concentration
10 mM) and incubated at 37
C for 1 h.
5. Proteins are alkylated with IAA (final concentration 50 mM) at
room temperature for 45 min.
6. After protein reduction and alkylation, 1 mL of AuNPs is added
to the sample and pH is adjusted to 4.0 with 1 M of citric acid.
7. The samples are incubated for 2 h at 37
C in a
thermostatic bath.
8. AuNP–protein complexes are harvested by centrifugation at
8600 Â g for 40 min.
9. The pellet is washed twice with 1 mL of citrate/citric acid
buffer (pH 4.0) to remove unbound protein.
3.3 SDS Gels
1. Protein pellets are reconstituted in 24 μL of Milli-Q water and
are mixed with 6 μL of SDS-PAGE loading buffer in a final
volume of 30 μL.
2. The samples are denatured by heating at 100
C for 5 min and
loaded into a 10% acrylamide/bis-acrylamide, stacking
gel/12.5% acrylamide/bis-acrylamide running gel, of 1 mm
126
Maria del Pilar Chantada-Va ´ zquez et al.