Rapid Purification of His-tagged Proteins

HEK293-F-FreeStyle cells (Invitrogen, Grand Island, NY) were transfected with plasmids encoding His, His-Si4 or Si4 fusion proteins using the Freestyle MAX reagent (Invitrogen, Grand Island, NY) and incubated for 24–72 hrs in 8% CO₂. Cells were harvested and lysed by sonication (Sonifier 250, Branson, Danbury, CT). His-tag or His-Si4 fusion proteins were purified on Ni/NTA beads as previously described [30 (link)], with the exception that the Si4 tag was used to immobilize PK on silica NPs directly from the cell lysate (S1D and S1E Fig), providing a fast and simpler purification process.

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Cohen R., Lata J.P., Lee Y., Hernández J.C., Nishimura N., Schaffer C.B., Mukai C., Nelson J.L., Brangman S.A., Agrawal Y, & Travis A.J. (2015). Use of Tethered Enzymes as a Platform Technology for Rapid Analyte Detection. PLoS ONE, 10(11), e0142326.

Publication 2015

Freestyle MAX Sonifier 250

Cell Hek293 cells Immobilize Plasmids Proteins Silica

Corresponding Organization : Atkins (United States)

Other organizations : SUNY Upstate Medical University, Presbyterian Hospital, New York Hospital Queens, NewYork–Presbyterian Hospital

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Variable analysis

independent variables

Transfection of plasmids encoding His, His-Si4 or Si4 fusion proteins

dependent variables

Purification of His-tag or His-Si4 fusion proteins on Ni/NTA beads
Immobilization of PK on silica NPs directly from the cell lysate using the Si4 tag

control variables

Incubation of transfected cells for 24–72 hrs in 8% CO2
Harvesting and lysis of cells by sonication

controls

Positive control: Purification of His-tag or His-Si4 fusion proteins on Ni/NTA beads as previously described [30]
Negative control: Not explicitly mentioned

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