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heparin conjugated affigel beads  (Bio-Rad)
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Bio-Rad heparin conjugated affigel beads
Vitreous humor contains a gap junctional intercellular communication-promoting activity with properties of an FGF. 50 ng/ml FGF-2 in M199/BOTS or E10 chick vitreous humor diluted with 2.3 vol of M199/BOTS (30% VH) was incubated with heparin-conjugated <t>Affigel</t> beads in the presence of 0.1 M NaCl or 0.6 M NaCl as described in Materials and methods. The beads were pelleted, and after removal of the supernatant (heparin 0.1 M or 0.6 M NaCl unbound), FGF-like activity was eluted with 2.5 M NaCl (heparin 2.5 M NaCl eluate). The fractions were then brought to 0.15 M NaCl by repeated rounds of concentration and dilution with M199 medium. DCDMLs were cultured for 2 d in M199/BOTS with no additions (control), in M199/BOTS with unfractionated FGF-2 (50 ng/ml FGF-2 input), in unfractionated 30% VH (30% VH input), or with the indicated heparin-Affigel fraction. Gap junctional intercellular communication was assessed as described in the legend to A; only Lucifer yellow immunofluorescence is presented. In all cases, rhodamine-dextran was confined to a single row of cells immediately bordering the wound. Representative results from three independent experiments; similar results were obtained with vitreous body conditioned medium (not shown). Note that the heparin beads quantitatively bound the communication-promoting activity of even the highest concentration of FGF tolerated by lens cells (50 ng/ml). The values given underneath the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment. Only the values obtained for the 30% VH input and the heparin 2.5 M NaCl eluate were significantly higher than control ( P < 0.03). Bar, 50 μm.
Heparin Conjugated Affigel Beads, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Vitreous humor contains a gap junctional intercellular communication-promoting activity with properties of an FGF. 50 ng/ml FGF-2 in M199/BOTS or E10 chick vitreous humor diluted with 2.3 vol of M199/BOTS (30% VH) was incubated with heparin-conjugated Affigel beads in the presence of 0.1 M NaCl or 0.6 M NaCl as described in Materials and methods. The beads were pelleted, and after removal of the supernatant (heparin 0.1 M or 0.6 M NaCl unbound), FGF-like activity was eluted with 2.5 M NaCl (heparin 2.5 M NaCl eluate). The fractions were then brought to 0.15 M NaCl by repeated rounds of concentration and dilution with M199 medium. DCDMLs were cultured for 2 d in M199/BOTS with no additions (control), in M199/BOTS with unfractionated FGF-2 (50 ng/ml FGF-2 input), in unfractionated 30% VH (30% VH input), or with the indicated heparin-Affigel fraction. Gap junctional intercellular communication was assessed as described in the legend to A; only Lucifer yellow immunofluorescence is presented. In all cases, rhodamine-dextran was confined to a single row of cells immediately bordering the wound. Representative results from three independent experiments; similar results were obtained with vitreous body conditioned medium (not shown). Note that the heparin beads quantitatively bound the communication-promoting activity of even the highest concentration of FGF tolerated by lens cells (50 ng/ml). The values given underneath the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment. Only the values obtained for the 30% VH input and the heparin 2.5 M NaCl eluate were significantly higher than control ( P < 0.03). Bar, 50 μm.

Journal: The Journal of Cell Biology

Article Title: A novel role for FGF and extracellular signal–regulated kinase in gap junction–mediated intercellular communication in the lens

doi: 10.1083/jcb.200101057

Figure Lengend Snippet: Vitreous humor contains a gap junctional intercellular communication-promoting activity with properties of an FGF. 50 ng/ml FGF-2 in M199/BOTS or E10 chick vitreous humor diluted with 2.3 vol of M199/BOTS (30% VH) was incubated with heparin-conjugated Affigel beads in the presence of 0.1 M NaCl or 0.6 M NaCl as described in Materials and methods. The beads were pelleted, and after removal of the supernatant (heparin 0.1 M or 0.6 M NaCl unbound), FGF-like activity was eluted with 2.5 M NaCl (heparin 2.5 M NaCl eluate). The fractions were then brought to 0.15 M NaCl by repeated rounds of concentration and dilution with M199 medium. DCDMLs were cultured for 2 d in M199/BOTS with no additions (control), in M199/BOTS with unfractionated FGF-2 (50 ng/ml FGF-2 input), in unfractionated 30% VH (30% VH input), or with the indicated heparin-Affigel fraction. Gap junctional intercellular communication was assessed as described in the legend to A; only Lucifer yellow immunofluorescence is presented. In all cases, rhodamine-dextran was confined to a single row of cells immediately bordering the wound. Representative results from three independent experiments; similar results were obtained with vitreous body conditioned medium (not shown). Note that the heparin beads quantitatively bound the communication-promoting activity of even the highest concentration of FGF tolerated by lens cells (50 ng/ml). The values given underneath the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment. Only the values obtained for the 30% VH input and the heparin 2.5 M NaCl eluate were significantly higher than control ( P < 0.03). Bar, 50 μm.

Article Snippet: 1 ml of either vitreous humor diluted with 2.3 vol of M199 (30% VH/M199) or M199 containing 50 ng/ml of FGF-2 was mixed end-over-end with 0.1 ml of heparin-conjugated Affigel beads (Bio-Rad Laboratories) in the presence of either 0.1 M NaCl or 0.6 M NaCl for 2 h at 4°C.

Techniques: Activity Assay, Incubation, Concentration Assay, Cell Culture, Control, Immunofluorescence, Standard Deviation