Biology Reference
In-Depth Information
2. Loading step: add the protein extract from Subheading
3.2
,
step 3
to the resin and shake on a rocking platform for 1 h at
4 °C. Centrifuge at 1,000 ×
g
for 2 min and recover the super-
natant for subsequent analysis by SDS PAGE.
3. Washing step: Add 15 mL of binding buffer to the Con A resin
with the bound protein sample and mix thoroughly for 1 min.
Centrifuge and recover supernatant/fl ow through. Repeat this
step twice more for a total of three washes. The spectrophoto-
metric absorbance (OD 280 nm) value can be used to approxi-
mate the protein concentration in the supernatant and used as
an indication of when no more protein is being eluted from the
resin. In general, three washes are suffi cient to remove most of
the nonspecifi cally bound protein, but additional washes can
be used if signifi cant amounts of proteins are still being eluted
after three washes.
4. Elution step: Add 500
L of elution buffer to the resin and mix
for 1 min. Centrifuge at 1,000 ×
g
for 2 min and recover the
supernatant for analysis. Repeat this step twice more. The gly-
coproteins will be present in these eluted fractions.
μ
3.5 Concentration
and Dialysis
1. Pool the three fractions from the elution step and reduce the
volume of the sample by 50 % in a centrifugal concentrator
using a 5 kDa cutoff centrifugal concentrator (Amicon Ultra-
15, Millipore, Billerica MA). Next perform a solvent exchange
step by fi rst adding an equal volume of 100 mM ammonium
bicarbonate (e.g., 4 mL of glycoprotein extract plus 4 mL
100 mM ammonium bicarbonate) then again reducing the
volume by 50 % by centrifugation. Repeat this solvent exchange
step at least three times.
2. Lyophilize the fi nal sample containing the glycoproteins
extract and resuspend the sample in 300
L resuspension
buffer, vortex for 5 min and place in a sonicating water bath for
5 min.
3. Centrifuge the suspension at 13,000 ×
g
for 3 min. Recover the
supernatant and set aside 50
μ
L for subsequent protein quan-
tifi cation and analysis by SDS-PAGE, and store the remainder
at −80 °C if necessary.
μ
3.6 Protein Digestion
1. Mix an aliquot of the sample containing 100
g of glycopro-
tein with resuspension buffer to a fi nal volume of 200
μ
μ
L.
2. Add 1
L 2 M DDT (the fi nal concentration will be 10 mM)
and incubate for 1 h at room temperature. Do not heat the
samples above room temperature (
see
Note 4
).
3. Add 20
μ
L 257 mM iodoacetamide (the fi nal concentration
will be 25 mM) and incubate for 30 min in the dark at room
temperature.
μ
