Lectin blotting

Purified glycoprotein or crude glycoprotein samples from cells, etc.

Transfer buffer for protein blotting: 25 mM of Tris, 192 mM of glycine, and 10% methanol

The nitrocellulose membranes or polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Hercules, CA)

The filter papers (Whatman International Ltd., Kent, UK or Bio-Rad Laboratories)

Tris-buffered saline (TBS): 137 mM of NaCl, 2.68 mM of KCl, and 25 mM of Tris-HCl, pH 7.4

Phosphate-buffered saline (PBS): 137 mM of NaCl, 8.1 mM of Na₂HPO₄, 2.68 mM of KCl, and 1.47 mM of KH₂PO₄, pH 7.4

Washing buffer: 0.05% (w/v) Tween-20 in TBS (TBST) or PBS (PBST)

Blocking solution: 3% (w/v) bovine serum albumin (BSA) or 3% (w/v) skim milk in TBST or PBST

Biotinylated lectins and HRP-labeled Streptavidin or alkaline phosphatase (AP)-labeled Streptavidin

Chromogenic or luminescent visualization regent for detecting tagged lectin or antibody

Two detection methods: the chemiluminescent detection of HRP activity using the luminol reagent (PIERCE Kit) and the conventional colorimetric reaction of AP revealed by nitroblue tetrazolium/bromochloro-indolyl phosphate (NBT/BCIP)

SDS-PAGE electrophoresis apparatus (e.g., mini-slab PAGE apparatus AE-6500, ATTO, Tokyo, Japan)

Transfer apparatus (e.g., Trans-Blot® SD Semi-Dry Transfer Cell, Bio-Rad Laboratories)

Image analyzer for chemiluminescence (e.g., LAS-4000, Fujifilm, Tokyo, Japan)

SDS-PAGE or 2D SDS-PAGE electrophoresis

a.

Prepare the glycoprotein samples.

b.

Run on 5%–20% SDS-PAGE or 2D SDS-PAGE gel until dye front is 0.5 cm away from the bottom of the gel.

c.

Set up the transfer apparatus.

Protein-blotting procedures

a.

Prepare transfer buffer while the gel is running and degas for at least 1 h before transfer.

b.

Remove the gel plates carefully and transfer to the container containing the transfer buffer.

c.

Wet the transfer nitrocellulose membrane thoroughly in a small amount of methanol (Note 1).

d.

Wet the filter papers and the transfer membrane thoroughly in the transfer buffer.

e.

Place transfer membrane on the gel and ensure that there are no bubbles.

f.

Place the 5 wetted pieces of filter papers on the membrane and ensure that there are no bubbles.

g.

Place them on the transfer assembly.

h.

Connect the lead to the power source and transfer for 1 h.

Lectin-blotting procedures

a.

After transferring proteins from the gel to the membrane, remove the membrane and rinse briefly in TBS or water.

b.

Treat the membrane with 15 mL blocking solution for 1 h at room temperature with gentle agitation (Note 2).

c.

Remove the blocking buffer and incubate the membrane for 1–2 h in biotinylated lectin at a concentration of 1 μg/mL in the blocking solution with agitation at room temperature. Usually, 15 mL of the lectin solution is used for incubating a membrane of 9 × 9 cm (Note 3).

d.

Wash the membrane at least 4 times for 5 min each with TBST.

e.

Add Streptavidin-HRP or Streptavidin-AP diluted in the blocking solution for 1 h at room temperature with agitation.

f.

Wash the membrane at least 4 times for 5 min each with TBST and then rinse twice with TBS.

g.

Perform chemiluminescence detection of peroxidase activity with a SuperSignal West Pico Chemiluminescent kit according to the manufacturer’s instructions (PIERCE). Incubate the membrane for 1 min at room temperature without agitation.

h.

Drain excess chemiluminescent solution by holding the membrane vertically. Then wrap blots in plastic sheet (Saran Wrap), without introducing air bubbles.

i.

Detect the luminescence with an automatic developer or manually by electronic imaging systems. The exposure time of the blots depends on the number of target proteins on the membrane (Note 4).

Instead of nitrocellulose, PVDF membrane can be used for lectin blotting.

Generally, BSA or nonfat dried skim milk is used to block membranes. For lectin blotting, we have tested BSA, which has produced a high background. When low-fat dried skim milk was used, it gave a low background for colorimetric and chemiluminescent detections. If no glycoprotein signals were obtained on the blot after the chemiluminescent reaction, this could be attributed to the presence of biotin in the skim milk that competed with biotinylated lectin and avoided its binding to the sugar moieties of glycoproteins.

The blots should be immersed in a sufficient volume of solution to allow a good exchange of fluid over its entire surface. The use of plastic bags to incubate protein blots is not recommended because it often leads to uneven background and may cause areas without signal. Depending on the carbohydrate specificity of the lectin, it may be necessary to optimize working dilution. Notably, when the calcium-dependent lectins, such as C-type lectin, are used, PBS must be replaced by another buffer system, such as TBS or HEPES buffer. Then, calcium phosphate precipitation occurs.

The difference in the exposure time depends on the lectin used. For example, if the lectin detects most of the N-linked glycoproteins, an exposure for a few seconds may be sufficient. Here, a longer exposure time leads to a high background with no additional spot signal.