Brock R, Hamelers I H, Jovin T M
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Cytometry. 1999 Apr 1;35(4):353-62. doi: 10.1002/(sici)1097-0320(19990401)35:4<353::aid-cyto8>3.0.co;2-m.
The analysis of the subcellular distribution of proteins is essential for the understanding of processes such as signal transduction. In most cases, the parallel analysis of multiple components requires fixation and immunofluorescence labeling. Therefore, one has to ascertain that the fixation procedure preserves the in vivo protein distribution. Fusion proteins with the green fluorescent protein (GFP) are ideal tools for this purpose. However, one must consider specific aspects of the fluorophore formation or degradation, i.e. reactions that may interfere with the detection of GFP fusion proteins.
Fusion proteins of the epidermal growth factor receptor (EGFR) with GFP as well as free, soluble GFP stably or transiently expressed in adherent cultured cells served as test cases for comparing the distribution in vivo with that after fixation by conventional epifluorescence and laser scanning microscopy. Indirect immunofluorescence was employed to compare the distributions of the GFP signal and of the GFP polypeptide in the fusion protein.
Paraformaldehyde (PFA) fixation with subsequent mounting in the antifading agent Mowiol, but not in Tris- or HEPES buffered saline, led to a partial redistribution of the EGFR from the plasma membrane to the perinuclear region. The redistribution was confirmed with the GFP and EGFR immunofluorescence. The in vivo distribution in Mowiol mounted cells was preserved if cells were treated with a combined PFA/methanol fixation procedure, which also retained the fluorescence of soluble GFP. The anti-GFP antiserum was negative for the N-terminal fusion protein.
The combined PFA/methanol protocol is universally applicable for the fixation of transmembrane and soluble cytoplasmic proteins and preserves the fluorescence of GFP.
蛋白质亚细胞分布的分析对于理解信号转导等过程至关重要。在大多数情况下，对多个组分进行平行分析需要固定和免疫荧光标记。因此，必须确定固定程序能够保留体内蛋白质的分布。绿色荧光蛋白（GFP）融合蛋白是实现这一目的的理想工具。然而，必须考虑荧光团形成或降解的特定方面，即可能干扰GFP融合蛋白检测的反应。
以在贴壁培养细胞中稳定或瞬时表达的表皮生长因子受体（EGFR）与GFP的融合蛋白以及游离的可溶性GFP作为测试案例，通过传统落射荧光显微镜和激光扫描显微镜比较体内分布与固定后的分布。采用间接免疫荧光法比较融合蛋白中GFP信号和GFP多肽的分布。
用多聚甲醛（PFA）固定，随后封固于抗淬灭剂Mowiol中，但不封固于Tris或HEPES缓冲盐溶液中，导致EGFR从质膜部分重新分布到核周区域。GFP和EGFR免疫荧光证实了这种重新分布。如果用PFA/甲醇联合固定程序处理细胞，Mowiol封固细胞中的体内分布得以保留，该程序也保留了可溶性GFP的荧光。抗GFP抗血清对N端融合蛋白呈阴性。
PFA/甲醇联合方案普遍适用于跨膜蛋白和可溶性细胞质蛋白的固定，并保留GFP的荧光。
