Mar 20, 2026
Hia5-His6 AdMTase purification
- Jonas Demeulemeester1
- 1Laboratory of Integrative Cancer Genomics, VIB-KULeuven
- Aertslab
Protocol Citation: Jonas Demeulemeester 2026. Hia5-His6 AdMTase purification. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2d1nqg1y/v1
Manuscript citation:
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: March 19, 2026
Last Modified: March 20, 2026
Protocol Integer ID: 313591
Keywords: ASAPCRN, his6 admtase purification, hia5 protein, protocol for the purification, purification, hia5, same as the original protocol, original protocol, his6
Abstract
This is a protocol for the purification of Hia5 protein and is basically the same as the original protocol as described by Stergachis et al., Science (2020). The pET30a-Hia5-His6 plasmid that we used was kindly provided by Andrew B. Stergachis.
Materials
BL21 derivative T7 Express lysY/Iq E. coli strain (McrA-, McrBC-, EcoBr-m-, Mrr-) NEB C3013I
pET-EcoGI/Hia5/Btr192V/Hin1523
IPTG: 100 mM stock
Kanamycin (Kan) 1000x: 30 mg/ml
100% H2O
STE buffer: 100 mM NaCl
10 mM TrisHCl pH 7.5
0.1 mM EDTA
Lysis buffer: 50 mM TrisHCl, pH 8
300 mM NaCl
1 mM DTT
20 mM Imidazole (make 5M stock at pH 8)
0.5 % Triton-X100
10 % glycerol
1 mM PMSF (add right before use for resuspension & same amount again after resuspension)
1 U / 10 mL DNase
His-select Ni affinity gel (Ni-NTA)
Wash buffer: 50 mM TrisHCl, pH 8
300 mM NaCl
1 mM DTT
20 mM Imidazole
Elution buffer: 50 mM TrisHCl, pH 8
250 mM NaCl
1 mM DTT
250 mM Imidazole
10% glycerol
Dialysis buffer: 50 mM TrisHCl, pH 8
250 mM NaCl
1 mM DTT
10% glycerol
Troubleshooting
Method
Bacterial growth
overnight: 200 mL preculture (1x Kan) per 2 L final culture of bacterial cells
transfer preculture to final 2 L LB-medium (1x Kan)
grow until OD = 0.8-1.0, take 1mL sample for SDS-PAGE
cool culture on ice to RT
add IPTG to final concentration of 0.5 mM
4hours at 20°C; take 1 mL sample for SDS-PAGE
spin down cells 10 min at 4000 rpm and 4°C
resuspend pellet in 20 mL lysis buffer (add the additional stuff mentioned in buffer composition section)
flash freeze and store suspension at -20°C
Lysis
everything on ice
thaw resuspended pellet (add the additional stuff mentioned in buffer composition section)
sonicate cells on ice 6 x 30 sec with 30 sec pauses, check viscosity (take sample for SDS-PAGE)
spin down cell debris 30 min at 15000 rpm
transfer cleared lysate to column (samples of Pellet and SN for SDS-PAGE)
Purification
add 2 mL His-select Ni affinity gel (capacity up to 15 mg/mL) to a disposable column
wash: 10 mL AD
equilibrate: 20 mL wash buffer (discard)
load the cleared lysate onto the column, use slow flow-rate (use a needle if necessary, retain flow-through, sample for gel)
wash: 20 mL wash buffer (higher flow-rate, retain flow-through, sample for gel)
elute: 12 mL elution buffer, fractions of 1 mL (use slow flow-rate, sample the fractions and the sepharose)
Analysis and concentration
put samples on gel: non-induced, induced, lysate, pellet, supernatant, flow-through, wash, sepharose, elutions
pool peak fractions as assessed by gel 6 dot-blot
dialyze overnight against 100x dialysis buffer or use PD10 desalting column
analyze protein concentration with Bradford reagent / UV
- Hia5: Mr 33,771.44 Da; ε280 43,110 M-1 cm-1
- EcoGI: Mr 41,040.82 Da; ε280 72,225 M-1 cm-1
- Btr192V: Mr 37,789.01 Da; ε280 50,100 M-1 cm-1
- Hin1523: Mr 33,991 Da; ε280 44,600 M-1 cm-1
store at -80°C or -20°C for use
Protocol references
Stergachis AB, Debo BM, Haugen E, Churchman LS, Stamatoyannopoulos JA. Single-molecule regulatory architectures captured by chromatin fiber sequencing.
Acknowledgements
We are grateful to Andrew B. Stergachis for providing the Hia5 expression construct and the lab of Zeger Debyser (Molecular Virology and Drug Discovery Laboratory, KULeuven) for their assistance with recombinant protein purification.