BibTex format
@article{Ramlaul:2023:10.1016/j.jsb.2023.107999,
author = {Ramlaul, K and Feng, Z and Canavan, C and de, Martin Garrido N and Carreno, D and Crone, M and Jensen, K and Li, B and Barnett, H and Riglar, D and Freemont, P and Miller, D and Aylett, C},
doi = {10.1016/j.jsb.2023.107999},
journal = {Journal of Structural Biology},
pages = {1--12},
title = {A 3D-printed flow-cell for on-grid purification of electron microscopy samples directly from lysate},
url = {http://dx.doi.org/10.1016/j.jsb.2023.107999},
volume = {215},
year = {2023}
}
RIS format (EndNote, RefMan)
TY - JOUR
AB - While recent advances in cryo-EM, coupled with single particle analysis, have thepotential to allow structure determination in a near-native state from vanishingly few individualparticles, this vision has yet to be realised in practise. Requirements for particle numbers thatcurrently far exceed the theoretical lower limits, challenges with the practicalities of achievinghigh concentrations for difficult-to-produce samples, and inadequate sample-dependent imagingconditions, all result in significant bottlenecks preventing routine structure determination usingcryo-EM. Therefore, considerable efforts are being made to circumvent these bottlenecks bydeveloping affinity purification of samples on-grid; at once obviating the need to produce largeamounts of protein, as well as more directly controlling the variable, and sample-dependent,process of grid preparation.In this proof-of-concept study, we demonstrate a further practical step towards thisparadigm, developing a 3D-printable flow-cell device to allow on-grid affinity purification fromraw inputs such as whole cell lysates, using graphene oxide-based affinity grids. Our flow-celldevice can be interfaced directly with routinely-used laboratory equipment such as liquidchromatographs, or peristaltic pumps, fitted with standard chromatographic (1/16”) connectors,and can be used to allow binding of samples to affinity grids in a controlled environment priorto the extensive washing required to remove impurities. Furthermore, by designing a devicewhich can be 3D printed and coupled to routinely used laboratory equipment, we hope toincrease the accessibility of the techniques presented herein to researchers working towardssingle-particle macromolecular structures.
AU - Ramlaul,K
AU - Feng,Z
AU - Canavan,C
AU - de,Martin Garrido N
AU - Carreno,D
AU - Crone,M
AU - Jensen,K
AU - Li,B
AU - Barnett,H
AU - Riglar,D
AU - Freemont,P
AU - Miller,D
AU - Aylett,C
DO - 10.1016/j.jsb.2023.107999
EP - 12
PY - 2023///
SN - 1047-8477
SP - 1
TI - A 3D-printed flow-cell for on-grid purification of electron microscopy samples directly from lysate
T2 - Journal of Structural Biology
UR - http://dx.doi.org/10.1016/j.jsb.2023.107999
UR - https://www.sciencedirect.com/science/article/pii/S104784772300062X
VL - 215
ER -