Protein Protocols Protein Protocols

Glycoprotein Detection 763
Schiff’s reagent is corrosive and slightly toxic and a very dilute solution will stain anything
with oxidized carbohydrates a pink-purple color Note: wear gloves and protective
clothing when using this solution and washing it out of the gel/blot.
4. Solution C: 50% (v/v) Ethanol.
5. Solution D: 0.5% (w/v) Sodium metabisulfite in 10 mM HCl.
6. Solution E: 7.5% (v/v) Acetic acid–5% (v/v) methanol in distilled water.
Solutions A, B, and D should be made up freshly.
2.2. DIG–Anti-DIG AP Labeling
1. Buffer A (TBS): 50 mM Tris-HCl, pH 7.5, 150 mM NaCl.
2. Buffer B: 100 mM Sodium acetate, pH 5.5.
3. Buffer C: 100 mM Tris-HCl, 50 mM MgCl2, 100 mM NaCl.
4. Buffer D: 250 mM Tris-HCl, pH 6.8, 8% (w/v) sodium dodecyl sulfate (SDS), 40% (v/v)
glycerol, 20% (v/v) 2-mercaptoethanol, bromophenol blue as tracking dye.
5. Buffer E: 50 mM potassium phosphate, 150 mM NaCl, pH 6.5 (PBS).
6. Blocking reagent: A fraction of milk proteins that are low in glycoproteins. 0.5g sample is
dissolved in 100 mL of buffer A. The solution should be heated to 60°C with stirring; the
solution will remain turbid. Allow the solution to cool before immersing the membrane.
7. DIG Glycan Detection Kit (Roche Diagnostics, Basel, Switzerland) containing:
a. Solution 1: 10 mM sodium metaperiodate in buffer B.
b. Solution 2: 3.3 mg/mL of sodium metabisulfite.
c. DIG-succinyl-ε-amidocaproic acid hydrazide.
d. Anti-DIG-AP: Polyclonal sheep anti-DIG Fab fragments, conjugated with AP (750 U/mL).
e. Solution 3: 75 mg/mL 4-nitroblue tetrazolium chloride dissolved in 70% (v/v)
dimethylformamide.
f. Solution 4: 50 mg/mL of 5-bromo-4-chloro-3-indolyl-phosphate dissolved in dimethylformamide.
Make sure that solutions 3 and 4 are still good, as after a few weeks the solutions may
begin to precipitate thus reducing the staining efficiency. Store these solutions (3 and 4)
in the dark.
2.3. DIG-Labeled Lectin Staining
1. 1% KOH.
2. Blocking reagent (Roche Diagnostics): A fraction of milk proteins that are low in glycoproteins.
A 0.5 g sample is dissolved in 100 mL of TBS. The solution should be heated to
60°C with stirring; the solution will remain turbid. Allow the solution to cool before
immersing the membrane. Other blockers such as skim milk powder, gelatin, or bovine
serum albumin (BSA) may lead to high background.
3. TBS: 50 mM Tris-HCl, pH 7.5, 150 mM NaCl.
4. TBS-Tween: TBS + 0.05% Tween 20.
5. Divalent cation stock solution: 0.1 M CaCl2, 0.1 M MgCl2, 0.1 M MnCl2. 6. DIG-labeled lectins (Roche Diagnostics): Sambucus sieboldiana agglutinin (SNA),
Maackia amurensis agglutinin (MAA), Arachis hypogaea (peanut) agglutinin (PNA), and
Datura stramonium agglutinin (DSA).
7. Part of DIG Glycan Detection Kit (Roche Diagnostics) comprising:
a. Anti-DIG-AP: Polyclonal sheep anti-DIG Fab fragments, conjugated with AP (750 U/mL)
b. Buffer C: Tris-100 mM HCl, 50 mM MgCl2, 100 mM NaCl.