Journal:

Article Title: Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

doi:

Figure Lengend Snippet: Scheme for the construction of the expression plasmids and the structures of their translation products. cDNAs for rat EGF and human bFGF were amplified by PCR from first-strand cDNAs prepared from the poly(A)+ RNA fraction of rat submaxillary gland and a human osteosarcoma cell line, respectively, using primers tagged with extra 5′ restriction sites. Each amplified cDNA was digested with restriction enzymes and then inserted into the BamHI–EcoRI site of pCHC302, giving pCHC302-EGF and pCHC302-bFGF, which express fusion proteins between GST and collagen-binding EGF (GST-CBEGF) and collagen-binding bFGF (GST-CBFGF), respectively. In the structure of GST-CBEGF (GST-CBFGF), amino acid residues derived from the pGEX-4T-2 plasmid vector are given in the single-letter code, and the thrombin-cleavage site is indicated by an arrow. The numbers in parentheses are the molecular weights of the domains.

Article Snippet: Affinity-purified anti-rat EGF antibodies ( 14 ), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, Heidelberg) were used as the primary antibodies.

Techniques: Expressing, Amplification, Binding Assay, Derivative Assay, Plasmid Preparation

Journal:

Article Title: Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

doi:

Figure Lengend Snippet: Purification profiles of CBEGF and CBFGF on SDS/PAGE. Samples at each purification step for CBEGF (lanes 2–5) and CBFGF (lanes 6–9), and the final preparation of human recombinant bFGF (lane 10) were electrophoretically separated in a SDS/13% polyacrylamide gel under reducing conditions and then stained with Coomassie brilliant blue R-250. Lane 1, molecular weight markers; lane 2, E. coli BL21/pCHC302-EGF crude extract; lanes 3 and 7, eluate from glutathione-Sepharose; lanes 4 and 8, thrombin digest of the eluate from glutathione-Sepharose; lane 5, eluate from Resource Q (purified CBEGF); lane 6, E. coli BL21/pCHC302-bFGF crude extract; lane 9, eluate from heparin-Sepharose (purified CBFGF); lane 10, purified human recombinant bFGF.

Article Snippet: Affinity-purified anti-rat EGF antibodies ( 14 ), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, Heidelberg) were used as the primary antibodies.

Techniques: Purification, SDS Page, Recombinant, Staining, Molecular Weight

Journal:

Article Title: Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

doi:

Figure Lengend Snippet: Dose–response curves for the growth factor activity of rat EGF, CBEGF, human bFGF, and CBFGF. BALB/c 3T3 A31 cells (2 × 104 cells in 2 ml of DMEM-2% calf serum) were inoculated onto 6-well multiwell plates, and 7 h later test samples were added. The cell number was determined with a Coulter counter after 4 days culture. The cell numbers in the absence of test samples and in the presence of 10% calf serum were 29,100 ± 1,600 and 239,600 ± 9,900, respectively. Each point represents the mean value ± SEM for triplicate experiments.

Article Snippet: Affinity-purified anti-rat EGF antibodies ( 14 ), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, Heidelberg) were used as the primary antibodies.

Techniques: Activity Assay

Journal:

Article Title: Collagen-binding growth factors: Production and characterization of functional fusion proteins having a collagen-binding domain

doi:

Figure Lengend Snippet: Detection of S-phase cells as BrdU incorporation and immunohistochemical staining of CBFGF in subcutaneous tissue of nude mice injected with CBFGF and human bFGF. The animals received an i.p. injection of BrdU (10 mg/100 g body weight) 24 h before death. Immunolocalization was performed on 5-μm paraffin sections by the streptoavidin-biotin-alkaline phosphatase complex technique using anti-BrdU mAbs (a-d) or anti-human bFGF antibodies (e and f) as the primary antibodies. (a, e, and f) 5 days after injection of CBFGF (50 μg). (b) 7 days after injection of CBFGF. (c and d) 5 days and 7 days, respectively, after injection of human bFGF (20 μg). (Bars, 100 μm.)

Article Snippet: Affinity-purified anti-rat EGF antibodies ( 14 ), anti-human bFGF antibodies (R & D Systems) and anti-BrdU mAbs (Progen, Heidelberg) were used as the primary antibodies.

Techniques: BrdU Incorporation Assay, Immunohistochemical staining, Staining, Injection