American Society of Hematology

Figure 3.

$Figure 3. Identification of the platelet protein recognized by the cloned human antiplatelet Abs by Western blotting and mass spectrometry. (A) Platelet lysate was fractionated by size exclusion chromatography, and fractions 14, 15, and 16 were separated by SDS-PAGE. The gel was divided and either silver-stained (Ai) or analyzed by Western blotting with Fab D4 (Aii; 2μg/mL), the negative control anti-gp120 Fab b12 (Aiii; 2 μg/mL), or murine antitalin IgG Ab TA205 (Aiv; 5 μg/mL). Arrows 1 to 3 indicate the positions of the protein bands (70, 47, and 42 kDa) specifically recognized by Fab D4 in Western blotting and present on silver-stained gel. Protein bands 1 to 3 were separately excised from the silver-stained gel, destained, and analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), identifying the talin head domain as the antigen recognized by antiplatelet Fab D4. Western blots of highly purified talin digested by calpain II (Av, lane 2) or not (Av, lane 1) stained with murine anti-human talin-H mAb TA205 (Av) were used as a reference. (B) Mass spectrum obtained from the MALDI-TOF analysis of the peptides derived from the 47-kDa protein band (band 2). (C) Talin-derived peptides. DA indicates Dalton.$

Identification of the platelet protein recognized by the cloned human antiplatelet Abs by Western blotting and mass spectrometry. (A) Platelet lysate was fractionated by size exclusion chromatography, and fractions 14, 15, and 16 were separated by SDS-PAGE. The gel was divided and either silver-stained (Ai) or analyzed by Western blotting with Fab D4 (Aii; 2μg/mL), the negative control anti-gp120 Fab b12 (Aiii; 2 μg/mL), or murine antitalin IgG Ab TA205 (Aiv; 5 μg/mL). Arrows 1 to 3 indicate the positions of the protein bands (70, 47, and 42 kDa) specifically recognized by Fab D4 in Western blotting and present on silver-stained gel. Protein bands 1 to 3 were separately excised from the silver-stained gel, destained, and analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), identifying the talin head domain as the antigen recognized by antiplatelet Fab D4. Western blots of highly purified talin digested by calpain II (Av, lane 2) or not (Av, lane 1) stained with murine anti-human talin-H mAb TA205 (Av) were used as a reference. (B) Mass spectrum obtained from the MALDI-TOF analysis of the peptides derived from the 47-kDa protein band (band 2). (C) Talin-derived peptides. DA indicates Dalton.

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