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. Author manuscript; available in PMC: 2012 Oct 3.
Published in final edited form as: Proteomics. 2011 Nov 23;11(24):4660–4676. doi: 10.1002/pmic.201100058

Figure 3.

Figure 3

SDS-PAGE of recombinant α1(XI) NTD, cartilage extract, and affinity selected proteins. Proteins were separated by SDS-PAGE and stained with Coomassie Safe Blue. (A) Molecular weight markers (lane 1), purified recombinant α1(XI) NTD protein (lane 2), proteins bound to nonderivatized column resin indicating nonspecific binding (lane 3), and total protein extract (lane 4). Bands or regions of the gel from lane 4 were manually excised, trypsinized, and analyzed by mass spectrometry to determine identity. For each region of the gel, the following proteins were identified: In the 240/230Mr range: biglycan, collagen α1(XI), actin, aggrecan, perlecan, collagen α1(XII), decorin, collagen α1(I), epiphycan, and collagen α1(XIV); in the 210Mr range: collagen α1(XIV), collagen α1(XII), biglycan, and tenascin-C; in the 180Mr range:thrombospondin-1, biglycan, collagen α1(XIV), perlecan, biglycan, collagen α1(XII), nidogen2,tenascin-C, collagen α1(I), collagen α1(XII), and fibromodulin; in the 160Mr range: collagen α1(II), biglycan, thrombospondin-1, fibromodulin, and collagen α1(I); in the 140Mr range: biglycan and COMP; in the 130Mr range: COMP and thrombospondin-1; in the 120Mr range: COMP, collagen α1(IX), biglycan, collagen α1(XIV), fibromodulin, thrombospondin-1, and matrilin-3; in the 100Mr range: biglycan, actinin alpha-4,fibromodulin, collagen α1(IX), matrilin-3, collagen α1(XIV), thrombospondin-1, collagen α1(II), nucleolin, pyruvate kinase, ovalbumin, and elongation-factor 2; in the 75Mr range: fibromodulin, lysyl hydroxylase, biglycan, collagen α1(II), thrombospondin-1, protein disulfide isomerase A4, and heat shock protein (GRP78); in the 60Mr range: CMP, protein disulfide isomerase A4, heat shock protein (GRP78), transferrin, semenogelin/inhibin, protein disulfide isomerase A1, actin, pyruvate kinase M2, collagens, phosphoglycerate kinase, vitrin, calreticulin, chondrocalcin, protein disulfide isomerase A5, and PARP; in the 48Mr range: actin, phosphoglycerate kinase, collagen-binding protein, and vitrin; in the 45Mr range: collagen α1(XI) NTD, fructose bisphosphate aldolase, CMP, and cartilage link protein; in the 40Mr range: chondroadherin, chondrocalcin, CMP, fructose bisphosphate aldolase, cartilage link protein, actin, and collagen α1(XI) NTD; in the 38Mr range: annexin A1, annexin A2, annexin A5, chondrocalcin, chondroadherin, lactate dehydrogenase B, lactate dehydrogenase A, collagen α1(XI) NTD, PARP, CMP, thrombosponin-1, fructose bisphosphate aldolase, and actin; in the 35Mr range: chondrocalcin, chondroadherin, annexin A1, annexin A2, annexin A5, CMP, thrombospondin-1, PARP, actin, lactate dehydrogenase A, and collagen α1(XI) NTD; in the 32Mr range: chondrocalcin, chondroadherin, annexin A1, annexin A2, annexin A5, thrombospondin-1, lactate dehydrogenase A, collagen α1(XI) NTD, and ANP32B; in the 28Mr range: collagen α1(I), PARP, chondrocalcin, CMP, thrombospondin-1, and collagen α1(XI) NTD, in the 25Mr range: PARP and CMP; in the 22Mr range: keratin; in the 20Mr range: lectin, histone 2A, histone 2B, collagen α1(XI) NTD; and in the 18Mr range: hemoglobin.

(B). Cartilage proteins that interact with the NTD of collagen α1(XI). SDS-PAGE stained with Coomassie Safe Blue. Molecular weight markers (lane 1), proteins selected by affinity chromatography (lane 2). Bands or regions of the gel indicated by letters a through p on the right-hand side of gel were manually excised, trypsinized, and analyzed by mass spectrometry. Proteins identified in the specific locations a through p were as follows: a) (240Mr): biglycan; b) (180Mr) collagen α1(XIV), thrombospondin-1, perlecan, biglycan, and collagen α1(XII); c) (160Mr)collagen α1(XII), biglycan, thrombospondin-1, and fibromodulin; d) (130Mr) thrombospondin-1; e) (120Mr) COMP, collagen α1(IX), biglycan, collagen α1(XIV), fibromodulin, thrombospondin-1, and matrilin-3; f) (100Mr) biglycan, actinin alpha-4, fibromodulin, collagen α1(IX), PARP, matrilin-3, collagen α1(XIV),thrombospondin-1, and collagen α1(XII) ; g) (90Mr) nucleolin and fibromodulin; h) (75Mr) fibromodulin, lysyl hydroxylase 1, biglycan, collagen α1(XII), thrombospondin-1, protein disulfide A4, transferrin, and heat shock protein 70; i) (60Mr) CMP, protein disulfide A3, fibromodulin, chondrocalcin, PARP, and calreticulin; j) (45Mr) CMP; k) (40Mr) CMP and 1,6 fructose bisphosphate aldolase; l) (38Mr) chondrocalcin, annexin A2, annexin A1, annexin A5, CMP, PARP, thrombospondin-1, 1,6 fructose bisphosphate and actin; m) (36Mr) chondrocalcin, annexin A5, annexin A1, annexin A2, CMP. thrombospondin -1, PARP, and actin; n) (34Mr) chondrocalcin, acidic leucine-rich nuclear phosphoprotein 32 family member B, annexin A1, annexin A2, annexin A5, collagen α1(XI) NTD, chondroadherin, lactate dehydrogenase A, thrombospondin-1, PARP, and CMP; o) (29Mr) PARP, chondrocalcin, CMP, thrombospondin-1, 1,6- fructose bisphosphate aldolase, and actin; p) (27Mr): PARP, thrombospondin-1, and collagen α1(XI) NTD.

(C) Grid depicting proteins identified as a function of location on gel. Apparent molecular weight is indicated on the vertical axis and protein identity is indicated along the horizontal axis.