Resources Selection Guide: Gator Probes for His-tagged Protein Capture Find the best probe for your His-capture assay When working with His-tagged proteins, not all capture strategies are equal. Crude lysate introduces non-specific binding, some proteins have less-accessible tags, and high throughput assays demand faster binding steps and sensitivity. Gator® Bio offers three His-tag biosensor capture chemistries, each built for different assay needs. Choosing the right one depends on your sample matrix, your buffer composition, the affinity you need to resolve, and the speed required for your assay. Access the full details and recommendations by downloading the Selection Guide PDF: Download Selection Guide Recommendations based on sample characteristics Sample/Assay CharacteristicsAnti-His(PN: 160009)Ni-NTA(PN: 160016)Anti-His (HIS) XT(PN: 160050)Crude samples or complex matrices✔✔Buffers with reducing agents or chelators✔✔Picomolar affinity measurements✔Small analytes (< 15 kDa) or weak interactions✔Analysis of Fc receptor binding✔✔Sample-limited applications✔Standardized, high-throughput workflows in clean buffers✔✔Table 1: Recommended His-capture probes for common sample and assay needs The right choice usually comes down to two questions: how clean is the sample, and how sensitive does the measurement need to be. Anti-His and Anti-His XT both tolerate certain crude lysates and buffers containing reducing agents or chelators, conditions that will strip a Ni-NTA surface, because nickel-nitrilotriacetic acid coordination depends on an intact metal-chelate bond. Ni-NTA remains a strong choice for purified samples in standardized, high-throughput workflows where buffer composition is controlled. Anti-His XT is ideal for picomolar affinity work, small analytes under 15 kDa, weak interactions, and sample-limited applications where you can’t afford to use much material. Key probe features to consider FeatureAnti-His(PN: 160009)Ni-NTA(PN: 160016)Anti-His (HIS) XT(PN: 160050)Capture SurfaceMonoclonal AntibodyNi-NTA CoordinationAI-Designed NovoBody™ApplicationsQ / K / EPQ / K / EPQ / K / EPQuantitation Dynamic Range0.25 – 500 µg/ml0.1 – 1000 µg/ml0.2 – 1000 µg/mlSample RequirementsHighModerateLowCompatible Sample MatrixPurified proteinsE coli lysateCHO LysateExpi293 LysatePurified proteinsPurified proteinsE coli lysateCHO lysateRegeneration / Reuse5-10 regenerations10+ regenerations10+ regenerationsKey StrengthsSpecific His-tag recognition; compatible with many buffersSimple, widely used capture chemistry; broad dynamic rangeHigher signal response per binding event, excellent ligand affinity, low ligand use, optimized to reduce avidityTable 2: Feature comparison between Gator Bio Anti-His, Ni-NTA, and Anti-His (HIS) XT probes Anti-His ProbesAnti-His captures His-tagged protein through a monoclonal antibody surface. It handles purified protein, E. coli lysate, CHO lysate, and Expi293 lysate, and supports quantitation, kinetics, and epitope binning across a dynamic range of 0.25 to 500 µg/mL. The surface regenerates 5 to 10 times using Gator Regen Buffer (Salt) (PN: 120012), and its antibody-based recognition keeps working in buffers that would disrupt a metal-chelate surface. Ni-NTA ProbesNi-NTA uses nickel-nitrilotriacetic acid coordination to capture His-tagged protein directly, without an antibody intermediate. It’s built for purified protein in clean buffer, with a quantitation dynamic range of 0.1 to 1000 µg/mL and 10 or more regeneration cycles using Gator Ni-NTA Regen Buffer and Ni-NTA Neutral Buffer, available together as the Ni-NTA Kit. It’s a straightforward, well-established capture chemistry for labs running standardized workflows where sample purity is already controlled upstream. Anti-His (HIS) XT ProbesAnti-His XT captures His-tagged protein using an AI-designed NovoBody™. It requires the least sample input of the three chemistries, supports a 0.2 to 1000 µg/mL dynamic range, and regenerates 10 or more times using Gator Regen Buffer pH 2.0 (no salt). The higher signal response per binding event, higher affinity His-tag capture and reduced avidity make it the choice for kinetics characterization (in particular picomolar affinity work), small analytes, and assays where sample volume is limited. See more details on HIS XT performance in our Application Note. Download the full selection guide here: Download Selection Guide FAQs What’s the difference between Anti-His, Ni-NTA, and Anti-His XT biosensors? Anti-His and Anti-His XT (HIS XT) both use protein-based affinity capture surfaces that tolerate crude lysates and chelator-containing buffers. Ni-NTA captures His-tagged protein through direct metal-chelate coordination and works best with purified samples in clean, standardized buffers. Which His-tag biosensor should I use for crude lysate samples? Anti-His and Anti-His XT (HIS XT) are both built for crude samples and complex matrices, including E. coli, CHO, and Expi293 lysates (see probe-specific recommendations in Table 2). Ni-NTA is recommended for purified proteins only. Which His-tag biosensor should I use for a high throughput cell-free capture workflow? Anti-His XT (HIS XT) has the fastest ligand-loading ability and the greatest sensitivity of the three, ensuring that hits are rapidly and reliably discovered. It also has reduced baseline drift, ensuring that analyte binding kinetics are accurate. Which biosensor measures picomolar affinity interactions? Anti-His XT (HIS XT) is the chemistry built for picomolar affinity measurements, along with small analytes under 15 kDa and other weak interactions. Can His-tag biosensors be reused after regeneration? Yes. Anti-His supports 5 to 10 regeneration cycles (sample and condition dependent). Ni-NTA and Anti-His XT both support 10 or more. Which His-tag biosensor works in buffers with reducing agents or chelators? Anti-His and Anti-His XT tolerate reducing agents and chelators. Ni-NTA does not, because its capture mechanism depends on an intact nickel-chelate bond that these compounds disrupt. How much sample do I need for each His-tag biosensor? Anti-His has the highest sample requirement, Ni-NTA is moderate, and Anti-His XT requires the least input of the three, making it the choice for sample-limited applications. Get in touch Still not sure which probe fits your application? Get in touch with us at [email protected]. Shop now Find the right biosensors for your research. Explore the Gator Bio catalog and shop biosensors for your biolayer interferometry assays. Browse complete biosensor catalog