PDF(Pubmed)
Abstract：
Protein-specific antibodies are essential for various aspects of protein research, including detection, purification, and characterization. When specific antibodies are unavailable, protein tagging is a useful alternative. Small epitope tags, typically less than 10 amino acids, are widely used in protein research due to the simple modification through PCR and reduced impact on the target protein\'s function compared to larger tags. The 2B8 epitope tag (RDPLPFFPP), reported by us in a previous study, has high specificity and sensitivity to the corresponding antibody. However, when attached to the C-terminus of the target protein in immunoprecipitation experiments, we observed a decrease in detection signal with reduced immunity and low protein recovery. This phenomenon was not unique to 2B8 and was also observed with the commercially available Myc tag. Our study revealed that C-terminal tagging of small epitope tags requires the addition of more than one extra amino acid to enhance (restore) antibody immunities. Moreover, among the amino acids we tested, serine was the best for the 2B8 tag. Our findings demonstrated that the interaction between a small epitope and a corresponding paratope of an antibody requires an extra amino acid at the C-terminus of the epitope. This result is important for researchers planning studies on target proteins using small epitope tags.
摘要：
蛋白质特异性抗体对于蛋白质研究的各个方面至关重要，包括检测，净化,和表征。当特异性抗体不可用时，蛋白质标记是一个有用的选择。小表位标签，通常少于10个氨基酸，由于通过PCR进行简单的修饰，并且与较大的标签相比，对靶蛋白功能的影响降低，因此被广泛用于蛋白质研究。2B8表位标签（RDPLPFFPP），我们在之前的研究中报道过，对相应的抗体具有较高的特异性和敏感性。然而,当在免疫沉淀实验中连接到目标蛋白的C末端时，我们观察到检测信号降低，免疫力降低，蛋白质回收率低。这种现象不是2B8所独有的,并且也用市售的Myc标签观察到。我们的研究表明，小表位标签的C末端标记需要添加一个以上的额外氨基酸以增强（恢复）抗体免疫力。此外，在我们测试的氨基酸中，丝氨酸对2B8标签最好。我们的发现表明，小表位与抗体的相应互补位之间的相互作用需要在表位的C末端有一个额外的氨基酸。该结果对于计划使用小表位标签对靶蛋白进行研究的研究人员很重要。
