Journal: Biochemistry

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65 †

doi: 10.1021/bi034002i

Figure Lengend Snippet: A Biotinylated Affinity-Labeling Agent for RBPs

Article Snippet: Biotinylated molecular mass markers (7, 14, 22, 31, 45, 66, 97, 116, and 200 kDa), two-color prestained markers (10, 15, 20, 25, 37, 50, 75, 100, 150, and 200 kDa), avidin-conjugated horseradish peroxidase, and a 10× Tris-buffered saline solution [20 mM Tris and 500 mM NaCl (pH 7.5)] were from Bio-Rad.

Techniques: Labeling

Journal: Biochemistry

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65 †

doi: 10.1021/bi034002i

Figure Lengend Snippet: 2D SDS–PAGE analysis of labeled RPE. (A) RPE proteome in 2D electrophoresis. Proteins were separated by isoelectric focusing (first dimension) and SDS–PAGE (second dimension). An immobilized pH gradient strip (pH 3 to 10, 13 cm) for IEF and a gradient gel (4 to 20%, 16 cm × 18 cm) for SDS–PAGE were used. Proteins were visualized by silver staining. (B) Biotin detection of labeled proteins. RPE was incubated with 1, at 10 μM, for 1 h at 4 °C. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL. Biotinylated molecular mass markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa) were loaded in the right-most lane. (C) Unlabeled control RPE. Proteins were transferred to a PVDF membrane and visualized by avidin-HRP/ECL.

Article Snippet: Biotinylated molecular mass markers (7, 14, 22, 31, 45, 66, 97, 116, and 200 kDa), two-color prestained markers (10, 15, 20, 25, 37, 50, 75, 100, 150, and 200 kDa), avidin-conjugated horseradish peroxidase, and a 10× Tris-buffered saline solution [20 mM Tris and 500 mM NaCl (pH 7.5)] were from Bio-Rad.

Techniques: SDS Page, Labeling, Two-Dimensional Gel Electrophoresis, Stripping Membranes, Silver Staining, Incubation, Avidin-Biotin Assay

Journal: Biochemistry

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65 †

doi: 10.1021/bi034002i

Figure Lengend Snippet: Time-dependent RPE labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after transferring proteins to the PVDF from the 4 to 12% SDS–PAGE gel: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, and 14 kDa); lane 2, RPE control; lane 3, RPE incubated with 1, at 5 μM, for 20 s at 4 °C; lane 4, RPE incubated with 1, at 5 μM, for 2 min at 4 °C; lane 5, RPE incubated with 1, at 5 μM, for 10 min at 4 °C; lane 6, RPE incubated with 1, at 5 μM, for 30 min at 4 °C; lane 7, RPE incubated with 1, at 5 μM, for 1 h at 4 °C; and lane 8, RPE incubated with 1, at 5 μM, for 3 h at 4 °C. (B) Time-dependent RPE labeling. The biotin signal is represented by a plot of volume vs time.

Article Snippet: Biotinylated molecular mass markers (7, 14, 22, 31, 45, 66, 97, 116, and 200 kDa), two-color prestained markers (10, 15, 20, 25, 37, 50, 75, 100, 150, and 200 kDa), avidin-conjugated horseradish peroxidase, and a 10× Tris-buffered saline solution [20 mM Tris and 500 mM NaCl (pH 7.5)] were from Bio-Rad.

Techniques: Labeling, Avidin-Biotin Assay, Transferring, SDS Page, Incubation

Journal: Biochemistry

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65 †

doi: 10.1021/bi034002i

Figure Lengend Snippet: Competition analysis of RBPs by preblocking and labeling. (A) Biotin detection analysis. Proteins were visualized by using avidin-HRP/ECL after SDS–PAGE on a 4 to 20% gradient gel. Proteins were transferred to a PVDF membrane: lane 1, biotinylated markers (200, 116, 97, 66, 45, 31, 22, 14, and 7 kDa); lane 2, RPE preblocked with 55 mM iodoacetamide for 1 h and then incubated with 1, at 5 μM, for 10 min at 4 °C; lane 3, RPE preblocked with 1 mM retinol for 1 h and then incubated with 1, at 5 μM, for 10 min at 4 °C; lane 4, RPE preincubated with 1 mM retinyl acetate for 1 h and then incubated with 1, at 5 μM, for 10 min at 4 °C; lane 5, RPE preincubated with 1 mM oleyl acetate and then incubated with 1, at 5 μM, for 10 min at 4 °C; lane 6, RPE preincubated with RBA (90 μM, 1 h) and then 1, at 5 μM, for 10 min at 4 °C; lane 7, RPE labeled with 1, at 5 μM, for 10 min at 4 °C; lane 8, control RPE; and lane 9, prestained molecular mass markers (177, 114, 81, 64, 50, 37, 26, 20, 15, and 8 kDa). (B) Relative intensity of RPE labeling compared to the control. The biotin signal is represented by volume in the graph.

Article Snippet: Biotinylated molecular mass markers (7, 14, 22, 31, 45, 66, 97, 116, and 200 kDa), two-color prestained markers (10, 15, 20, 25, 37, 50, 75, 100, 150, and 200 kDa), avidin-conjugated horseradish peroxidase, and a 10× Tris-buffered saline solution [20 mM Tris and 500 mM NaCl (pH 7.5)] were from Bio-Rad.

Techniques: Labeling, Avidin-Biotin Assay, SDS Page, Incubation

Journal: Biochemistry

Article Title: A Cleavable Affinity Biotinylating Agent Reveals a Retinoid Binding Role for RPE65 †

doi: 10.1021/bi034002i

Figure Lengend Snippet: Elution of biotinylated proteins from neutravidin–agarose beads. The neutravidin–agarose beads which contain the labeled RPE were treated with buffer at either pH 7.5 or 11: lane 1, pH 11 for 6 h; lane 2, pH 7.5 for 6 h; lane 3, pH 11 overnight; and lane 4, pH 7.5 overnight.

Article Snippet: Biotinylated molecular mass markers (7, 14, 22, 31, 45, 66, 97, 116, and 200 kDa), two-color prestained markers (10, 15, 20, 25, 37, 50, 75, 100, 150, and 200 kDa), avidin-conjugated horseradish peroxidase, and a 10× Tris-buffered saline solution [20 mM Tris and 500 mM NaCl (pH 7.5)] were from Bio-Rad.

Techniques: Labeling