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Figure 3 | BMC Biotechnology

Figure 3

From: Development of a recombinant antibody to target peptides and proteins to sialoadhesin-expressing macrophages

Figure 3

Development of fusion constructs rec41D3-V5 and rec41D3-eGFP and analysis of their internalization into pSn-expressing cells. (A) SDS-PAGE analysis of protein G purified rec41D3-V5 and rec41D3-eGFP constructs in comparison to unmodified rec41D3 (B) Western blot analysis of rec41D3-V5 and rec41D3-eGFP samples in comparison to unmodified rec41D3 using mouse immunoglobulin-specific goat polyclonal antibodies (GαM), a V5-specific mAb (αV5) and an eGFP-specific mAb (αGFP). (C) Confocal microscopical analysis of rec41D3-V5 and rec41D3-eGFP internalization in CHO-pSn cells and primary macrophages (PAM). Cells were incubated with rec41D3-V5 or rec41D3-eGFP for 1 hour at 37°C, fixed, permeabilized and stained with AF594-labelled goat-anti-mouse IgG1 in addition to FITC-labelled mouse anti-V5 (IgG2a) or rabbit anti-GFP in combination with FITC-labelled goat-anti-rabbit polyclonal antibodies respectively. Since the rec41D3-eGFP fluorescence was weak, the signal was enhanced to obtain good microscopical images. Images represent a single confocal z-section through the middle of the cell. M, protein marker; -, rec41D3; V5, rec41D3-V5; eGFP, rec41D3-eGFP. Scale bar: 5μm.

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