Biology Reference
In-Depth Information
3.2.2. Sample Binding
1.
Centrifuge the serum sample at 10,000 ×
g
for 10 min if
necessary. This is to ensure that the sample is free of precipitate
or aggregate proteins and/or lipids, which will affect the
binding of proteins to the beads.
2.
Add 200 mL of serum to the column. Replace the top cap and
tap the column to mix. Place the spin column on a rotary
shaker and incubate for 2 h for sample binding to take effect
(see Note 4).
3.2.3. Sample Wash
1.
Place the spin column in a capless collection tube. Centrifuge
at 1,000 ×
g
for 1 min. Discard the solution containing mainly
the unbound proteins.
2.
Replace the bottom cap and add 200 mL of wash buffer to the
beads. Replace the top cap and tap the column to mix.
3.
Wash the beads in the spin column by rotating on a rotary
shaker for 5 min.
4.
Place the spin column back to the capless collection tube.
Centrifuge at 1,000 ×
g
for 1 min and discard the solution
collected in the tube.
5.
Repeat steps 2-4 twice.
1.
Replace the bottom cap on the spin column. Add 200 mL of
deionized water. Replace the top cap and tap the column
to mix.
3.2.4. Sample Elution
2.
Place on the rotary shaker to shake for 1 min.
3.
Place the spin column in a capless collection tube. Centrifuge
at 1,000 ×
g
for 1 min and discard the solution collected in
the tube.
4.
Repeat steps 1-3.
5.
Replace and ensure that the bottom cap is tightly attached
onto the spin column. Add 20 mL of rehydrated elution reagent.
Replace the top cap and tap the column to mix.
6.
Incubate the column on the rotary shaker for 15 min, with
several rounds of light vortexing.
7.
Place the spin column in a clean collection tube. Centrifuge at
1,000 ×
g
for 1 min. Retain the eluate.
8.
Repeat steps 5-7 twice.
9.
Repeat steps 5-7 with a fi nal elution volume of 60 mL of rehy-
drated elution reagent.
10. Eluates may be pooled or analyzed individually (see Fig.
1
).
Store sample at −80°C or proceed with sample precipitation
using 2D Clean-up kit.
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