ChromoBlog

Why Spot-Tag can be used to purify any protein

Introduction

Proteins often have very different individual characteristics and requirements for manipulation. Not every purification tag in combination with the desired purification resin is suitable for every protein. Some proteins are sensitive towards wash or elution buffers, while others are difficult to purify because the selected affinity resin can be contaminated by host cell proteins. Taking these points into consideration, the selection of both the affinity tag and the affinity resin can have a strong impact on protein purity and yield, which is often dependent on the target protein. Requirements for an affinity resin comprise (i) high purity (minimal contaminations), (ii) high yield of the purified protein, and (iii) a broad buffer stability. Ideally, affinity tags should be (iv) inert and (v) short in order to not or minimally affect the purified protein.

New iST Nano-Trap kits for immunoprecipitation (IP) and sample preparation for mass spectrometry (MS) in just 4 easy steps:

Introduction

The Spot-Nanobody (green) binds to the Spot-Tag sequence motif PDRVRAVSHWSS. Upon binding, the Spot-Tag peptide is embedded on the surface of the Spot-Nanobody and becomes a β-sheet extension of the Spot-V_HH. Defined interactions of the Spot-Nanobody’s side chains to the Spot-peptide determine specificity. In addition, the Spot-peptide is clamped by two amino acid side chains of the Spot-Nanobody. This binding mechanism elucidates why the Spot-Nanobody binds with high affinity to the Spot-Tag.

You may have noticed the recent publication “A peptide tag-specific nanobody enables high-quality labeling for STORM imaging” of Virant et al (2018) in Nature Communications doi: 10.1038/s41467-018-03191-2, where for the first time a peptide-tag specific Nanobody was applied in dSTORM imaging: The authors have described and discussed the performance of a BC2 peptide-tag specific Nanobody to image BC2 peptide tag fusion proteins in super resolution microscopy, i.e. dSTORM. This Blog features that BC2 peptide-tag specific Nanobody, which ChromoTek markets under the names Spot-Label® and Spot-Trap®. In addition, this Blog figures the significantly improved version of the BC2 peptide -tag, which is called Spot-Tag®.