Biology Reference
In-Depth Information
by 3 mL of 50 % acetonitrile and 0.5 % formic acid. Equilibrate
with 9 mL of 0.1 % trifl uoroacetic acid. Load sample in 0.4 %
trifl uoroacetic acid. Wash/desalt with 9 mL of 0.1 % trifl uoro-
acetic acid. Wash (to remove trifl uoroacetic acid) with 900
L
of 0.5 % formic acid. Elute with 5 mL of 50 % acetonitrile and
0.5 % formic acid, and collect eluate in a 15 mL conical tube.
μ
6. Set the avidin column as recommended by the manufacturer:
mark the inlet and outlet ends of the cartridge (or mark with a
directional arrow) for future use. Use the same fl ow direction in
all runs to prevent particles that may accumulate at the car-
tridge inlet from clogging the outlet tubing. Insert the avidin
cartridge into the cartridge holder. Inject 2 mL of the Affi nity
Buffer-Elute using a syringe pump. Divert to waste (
see
Note
10
). Inject 2 mL of the Affi nity Buffer-Load. Divert to waste.
Neutralize each cation-exchange fraction by adding 500
L of
the Affi nity Buffer-Load. Check the pH using pH paper. If the
pH is not 7, adjust by adding more Affi nity Buffer-Load. Vortex
to mix and then centrifuge for a few seconds to bring all solu-
tion to the bottom of the tube. Remove an optional 1
μ
L pro-
cess-monitoring aliquot and label as “pre-avidin.” Label three
fraction-collection tubes: #1 (Flow-Through), #2 (Wash), and
#3 (Elute), then place in a rack. Slowly inject (~1 drop/s) the
neutralized fraction onto the avidin cartridge and collect the
fl ow-through into tube #1 (Flow-Through). Inject 500
μ
L of
Affi nity Buffer-Load onto the cartridge and continue to collect
in tube #1. (Keep tube #1 until you confi rm that loading on the
avidin cartridge is successful. If loading fails, you can repeat
loading using tube #1 after you troubleshoot the cause of the
loading failure. To reduce the salt concentration, inject 1 mL of
Affi nity Buffer-Wash 1. Divert the output to waste. To remove
nonspecifi cally bound peptides, inject 1 mL of Affi nity Buffer-
Wash 2. Collect the fi rst 500
μ
μ
L in tube #2. Divert the remain-
ing 500
L to waste. Inject 1 mL of water. Divert to waste. Fill
a syringe with 800
μ
L of the Affi nity Buffer-Elute. To elute the
labeled peptides, slowly inject (~1 drop/s) 50
μ
L of the Affi nity
Buffer-Elute and discard the eluate. Inject the remaining
750
μ
L of Affi nity Buffer-Elute and collect the eluate in tube #3
(Elute). Vortex to mix and then centrifuge for a few seconds to
bring all solution to the bottom of the tube. Remove an optional
1
μ
L process-monitoring aliquot after eluting from the avidin
cartridge, and label as “post-avidin.” If you have additional
cation-exchange fractions, repeat the steps described. Evaporate
each affi nity-eluted fraction to dryness in a centrifugal vacuum
concentrator. In a fresh tube, prepare the fi nal cleaving reagent
by combining Cleaving Reagent A and Cleaving Reagent B in a
95:5 ratio. You need ~90
μ
L of fi nal cleaving reagent per sam-
ple. Vortex to mix and then centrifuge for a few seconds to
bring all solution to the bottom of the tube. Transfer ~90
μ
μ
L of
