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Recombinant protein (200 pmol) was incubated with 5 µl of washed RBC in RPMI for 2.5 h at 4°C, washed and bound protein was visualized by Westernblot (α-GST mAb, dilution 1∶5000, Sigma; α-mouse mAb-ALP, dilution 1∶10000, Sigma).

By means of SFM, the lateral organization of membrane-bound protein was visualized showing micrometer-sized, sometimes interconnected STxB clusters (Figure 2B).

Bound proteins were visualized by autoradiography.

After washing the membranes, the bound proteins were visualized by a luminol detection system (Santa Cruz).

Bound proteins were visualized with the Odyssey Imaging System (Li-Cor Biosciences, Lincoln, NE, USA) and quantified with the Multigauge software (Fuji Film, Tokyo, Japan).

Following incubation with horseradish peroxidase-conjugated secondary antibody (Southern Biotechnology, Birmingham, AL, USA), bound proteins were visualized by chemiluminescence ECL (Amersham Biosciences, Freiburg, Germany).

After incubation with peroxidase-coupled anti-mouse/rabbit IgG (Santa Cruz Biotechnology) at 37°C for 2 h, bound proteins were visualized using ECL Piercee) and detected using BioImaging Systems (UVP Inc., Upland, CA).

(E, F ) HEK293T cells expressing FLAG-Aven and FLAG-AvenΔRGG were processed as in panel C, D except the bound proteins were visualized by immunoblotting with anti-FLAG antibodies.

After incubation with peroxidase-coupled antimouse IgG (Santa Cruz) at 37°C for 2 h, bound proteins were visualized using ECL (Pierce) and detected using a BioImaging System (UVP Inc., Upland, CA, USA).

After washing with TBS-T, bound proteins were visualized using anti-mouse or anti-rabbit IgG HRP-conjugated antibodies (GE; 1 40 000 in blocking buffer) and the SuperSignal West Femto chemiluminescence kit (Pierce).

GST-bound proteins were visualized by Coomassie Brilliant Blue (CBB) staining and the S-labeled proteins by fluorography.