Protocol 5

5. Immunostaining Thin Layer Chromatograms Of Glycolipids

John L. Magnani~GlycoTech Corporation, Rockville, Maryland 20850

Immunostaining thin layer chromatograms allows for very sensitive detection of functionally active carbohydrate ligands of protein receptors (1, 2). These carbohydrate structures are detected in glycolipids from complex mixtures of molecules extracted from the relevant target tissue. Some protein receptors that may be analyzed are: antibodies, chimeric Ig proteins, lectins, selectins, toxins, and other carbohydrate binding proteins. This method allows for the determination of the number, the size (Rf value) and the charge (combined with DEAE chromatography) of the carbohydrate ligand(s). Furthermore, this method aids in determining the method to use for purification of the glycolipid ligand and subsequently to monitor the purification process (3, 4). Contrary to glycoproteins, glycolipids contain one hapten oligosaccharide per molecule and therefore, once purified and structurally characterized, the glycolipid represents the functionally active carbohydrate sequence for the protein receptor under study.

Materials:

Chromatography Tank
Aluminum-backed HPTLC silica gel plates with fluorescence indicator

Glass microsyringe

Petri dishes (150 mm)

Glass microscope slides

Whatman #1 filter paper

Heat dryer

Spraying apparatus

Plastic squeeze bottle

Polyisobutyl methacrylate

Acetone

Chloroform
Methanol
Potassium chloride

Phosphate-buffered saline (0.01M sodium phosphate, 0.15M NaCl, pH 7.6)

Bovine serum albumin

Orcinol monohydrate

Resorcinol

Diaminobenzidine tetrahydrochloride (DAB)

Protocol:

Preparation of the Chromotography Tank

1. Add 150 ml of chloroform/ methanol/0.25%KCI (5:4:1) to a glass chromatography tank (25 cm x 27 cm x 10 cm) lined with filter paper and covered tightly.

2. Allow vapors in tank to equilibrate for at least 2 hours. For best results, prepare a new tank daily.

Preparation of the Silica Gel Plate

1. Cut the plate to the desired size using scissors. The optimum height should be 10 cm.

2. Draw a very light pencil line 1.5 cm parallel to the bottom of the plate being careful not to scratch the silica gel.

3. Using a glass microsyringe, spot the glycolipid sample along a 5 mm path on the pencil line. Separate these paths (lanes) by 2.5 mm.

4. Samples to be detected for sugar content by orcinol spray should be placed together on a portion of TLC plate separated from those samples to be immunostained.

Chromatography of the Silica Gel Plate

1. Using a pair of long forceps, grasp the top of the plate and place in the tank oriented with the spotted sample just above the level of the solvent. Allow the top edge to lean against the rear of the developing chamber.

2. Cover the tank tightly and allow the plate to remain undisturbed until the ascending solvent line reaches the top of the plate (approx. 30-45 minutes).

3. Remove the TLC plate by grasping the top edge with forceps and allow the plate to thoroughly air dry under chemical fume hood.

Staining the Standards for Carbohydrate

1. Cut off the portion of the TLC plate containing the glycolipid standards.

2. In a chemical fume hood, spray the plate briefly with 0.1% orcinol dissolved in 5% H₂SO₄.

3. Immediately place in a pre-warmed oven at 120�C for approximately 5-10 minutes.

4. If staining is weak, steps 2 and 3 may be repeated.

Preparation of the Chromatographed Plate for Immunostaining

1. Spot 2�l of positive (carbohydrate-binding protein) and negative (BSA) controls (0.5 mg/ml) at the bottom of the TLC plate and allow to completely air dry.

2. Soak the TLC plate for 90 seconds in a glass Petri dish (150 mm) containing a 0.1% solution of polyisobutylmethacrylate beads predissolved in acetone. For best results, the plate should enter the solution quickly, long side first, at a 45 degree angle.

3. Remove the plate and stand upright under a chemical fume hood to completely air dry.

4. Measure the surface area of the TLC plate.

5. Spray the TLC plate vigorously with PBS containing 1% BSA and then submerge immediately in the same solution in Petri dish for 1 hr.

Immunostaining With the Primary Carbohydrate-Binding Protein

1. Dilute the carbohydrate-binding protein (e.g. antibody) to 10�g/ml in PBS containing 1% BSA. The amount to be overlaid is 65 �l/cm² of TLC plate surface.

2. Prepare a pedestal slightly smaller than the TLC plate with glass microscope slides in the bottom of a Petri dish.

3. Remove the TLC plate from the PBS-1% BSA, drain, and place horizontally, silica gel side up, atop the glass slide pedestal. Do not let the plate dry during this and any following steps until the end of the protocol.

4. Using a pipette, gently cover the TLC plate evenly with the protein solution making sure that the liquid maintains its level atop the plate by surface tension.

5. Cover the Petri dish and allow the overlay to incubate for 2 hrs. at room temperature.

Detection of Bound Protein Using a Secondary Peroxidase-Labeled Anti-Protein

1. Dilute the secondary peroxidase-labeled anti-protein (e.g. peroxidase-labeled goat anti-mouse 1 g) to 1 �g/ml in PBS containing 1% BSA.

2. Thoroughly and gently wash the TLC plate with a squeeze bottle containing PBS over an open sink.

3. Lay the plate across a fresh glass pedestal as above and using a pipette, gently cover the plate evenly with the peroxidase-labeled anti-protein solution prepared in step 1.

4. Cover the dish and incubate for 1 hr. at room temperature.

Addition of Colorimetric Enzyme Substrate

1. Prepare fresh DAB solution as a substrate for horseradish peroxidase as described by the manufacturers instructions.

2. Thoroughly and gently wash the TLC plate with a squeeze bottle containing PBS over an open sink.

3. Overlay the TLC plate with the DAB substrate solution and incubate until the positive controls appear and before the background staining becomes too intense (about 5-10 mins.).

4. Wash extensively with distilled H₂O to stop the reaction and dry the plate under a stream of warm air from a heat dryer.

References:

(1) Magnani, J.L., Smith, D.F. and Ginsburg, V. (1980) Detection of gangliosides that bind cholera toxin: direct binding of ¹²⁵I-labeled toxin to thin-layer chromatograms. Anal. Biochem. 109: 399-402.

(2) Magnani, J.L., Brockhaus, M., Smith, D.F., and Ginsburg, V. (1982) Detection of glycolipid ligands by direct binding of carbohydrate-binding proteins to thin-layer chromatograms. Methods in Enzymology 83: 235-241.

(3) Magnani, J.L., Spitalnik, S.L., and Ginsburg, V. (1987) Antibodies against cell surface carbohydrates: Determination of antigen structure. Methods in Enzymology 138: 195-207.

(4) Magnani, J.L., Nilsson, B., Brockhaus, M., Zoph, D., Steplewski., Z., Koprowski, H., and Ginsburg, V.(1982) A monoclonal antibody-defined antigen associated with gastrointestinal cancer is a ganglioside containing sialylated lacto-N-fucopentaose II. J. Biol. Chem. 257: 14365-14369.