Jun 26, 2025
Version 2
Direct In Vitro Pulldowns of Hexahistidine-Tagged Proteins and Peptides with Interactors V.2
- Dieter Waschbuesch1,
- Amir R Khan2
- 1School of Biochemistry and Immunology, Trinity Biomedical Sciences, Institute Trinity College Dublin, Dublin 2, Ireland;
- 2School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland
- AKhanLab
Protocol Citation: Dieter Waschbuesch, Amir R Khan 2025. Direct In Vitro Pulldowns of Hexahistidine-Tagged Proteins and Peptides with Interactors. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpqk91lzp/v2Version created by Amir Khan
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 24, 2025
Last Modified: June 26, 2025
Protocol Integer ID: 220868
Keywords: ASAPCRN, pull-down, pulldown, in-vitro pulldown, direct pulldown, untagged proteins as prey, peptides with interactor, tagged protein, tagged peptide, untagged protein, direct in vitro pulldowns of hexahistidine, peptide, protein, strong two protein, purified protein, molar amounts of protein, direct in vitro pulldown, hexahistidine, known interactor, interactor, direct pulldown, direct binding
Funders Acknowledgements:
Research Ireland
Grant ID: 20/FFP-A/8446
Abstract
Direct pulldowns of two purified proteins are a standard tool to determine whether they bind directly to each other. Mutational analyses allow us to find out which residues are involved in the direct binding of known interactors. By using defined molar amounts of proteins, it is even possible to roughly estimate how strong two proteins interact with each other. Here, we describe a protocol that uses hexahistidine-tagged peptides and proteins as bait and untagged proteins as prey in vitro, with subsequent pulldown by Ni2+-agarose beads.
Materials
Consumables
- 1. 5 ml Reaction tubes (Greiner, 616201)
Reagents
- Purified bait and prey proteins/peptides
- Nickel-NTA agarose resin (e.g., Thermo Fisher R90110, etc.) equilibrated in binding buffer
- Binding buffer (i.e., 20 mM Tris pH 8.0 at 4°C, 300 mM NaCl, 10 mM β-Mercaptoethanol, 20 mM Imidazole)
Note
The amount of Imidazole can be varied depending on the proteins, as some are more or less sticky to the Nickel-agarose resin. In the vast majority of experiments, the above-mentioned buffer worked perfectly fine, providing a clean background in negative (=no bait) controls.
- Elution buffer (20 mM Tris pH 8.0 at 4°C, 300 mM NaCl, 200 mM Imidazole)
- Coomassie Staining solution (e.g., as described here: https://app.jove.com/t/1350/staining-proteins-gels-with-coomassie-g-250-without-organic-solvent)
Equipment
- Overhead rotator (LD-79, LABINCO BV)
- SDS-PAGE gels and electrophoresis system (ATTO)
- Tabletop microcentrifuge (Ohaus or VWR MicroStar 12)
Troubleshooting
Protocol
25m
Pipetting scheme and general considerations
Perform all steps on ice or in the cold room.
Determine the concentrations of your proteins or peptides.
Note
Depending on the molecular weight of the protein/peptide, a concentration between 1-5 mg/ml is ideal.
Typically, 10 µM bait and prey in 1 ml, but according to the amount and availability of the protein, lower concentrations and/or volume work fine.
Note
Volumes below 500 µl are possible but not recommended
Always include a no-bait control to confirm that the non-His-tagged protein does not bind to the resin.
Triplicates are better than duplicates, which are better than just one condition.
Formula to calculate the molar concentration:
Here c = concentration in gram/liter (or, mg/ml), MW = Molecular Weight (gram/ mol)
Example:
- Bait: c=2 mg/ml, MW=20000 g/mol → 100 µM. 10 µM in 1 ml = 100 µl
- Prey: 3.6 mg/ml, MW=40000 g/mol → 90 µM. 10 µM in 1 ml = 111 µl
Table 1: Example pipetting scheme
| 1 | 2 | 3 | 4 | 5 | 6 | ||
| Bait | 100µl | 100µl | 100µl | 100µl | 100µl | 100µl | |
| Prey | 111µl | 111µl | 111µl | ||||
| Buffer | 850µl | 850µl | 850µl | 739µl | 739µl | 739µl |
Pulldown
Pipette your binding buffer, bait, and prey protein according to the pipetting scheme.
Add 50 µl of 50% slurry Ni-NTA agarose beads equilibrated in binding buffer to the Eppendorf tubes.
Incubate in an overhead rotator for 15 minutes. If no rotator is available, invert tubes every minute by hand.
15m
Spin down samples at 1000 x g for 2 minutes.
2m
Remove supernatant. If required, collect a 15 µl sample of the 'unbound' fraction (U).
Resuspend in 1 ml of binding buffer for ~1 minute.
1m
Spin down samples at 1000 x g for 2 minutes.
2m
Remove supernatant and wash cells with binding buffer.
Repeat the wash step two times.
Remove supernatant carefully and completely after the last wash.
Note
Best done in two steps: remove most of the supernatant with a 1 ml pipette, wait ~1 minute, then remove the remaining supernatant with a 200 µl pipette, so that the Ni-agarose resin falls dry.
Elute proteins with 50 µl of elution buffer by vortexing briefly.
Spin down at full speed in an Eppendorf centrifuge.
Mix 36 µl supernatant with 12 µl 4x SDS-PAGE loading buffer.
Incubate at 95°C for 5 minutes.
5m
Vortex, spin down samples.
Make input samples: Dilute 40 µg of your bait and prey proteins in 100 µl (75 µl protein sample + dH2O and 25 µl 4x SDS-PAGE loading buffer). A load of 5 µl per lane contains 2 µg of protein, which gives a well-visible band in a conventional SDS-PAGE (Laemmli).
Note
Samples can now be stored at -20°C or directly be analysed by SDS-PAGE.