Production of Recombinant Semaphorin 4D

Recombinant Semaphorin 4D was produced as a secreted protein fused with AP. To produce recombinant Semaphorin, human embryonic kidney HEK293T cells were transfected with Sema4D-AP in pcDNA3 vector47 (link), using Fugene 6 reagent according to the manufacturer’s protocol (Promega) The supernatant of transfected cells was collected, filtered and concentrated 10-fold by Spin-X UF concentrators with a 100kDa cutoff (Millipore). Protein concentration was normalized to a range of 3–6 nM by measurement of the AP-activity using Alkaline Phosphatase Colorimetric Assay kit (Abcam).

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Azzarelli R., Pacary E., Garg R., Garcez P., van den Berg D., Riou P., Ridley A.J., Friedel R.H., Parsons M, & Guillemot F. (2014). An antagonistic interaction between PlexinB2 and Rnd3 controls RhoA activity and cortical neuron migration. Nature Communications, 5, 3405.

Publication 2014

Fugene 6 reagent Spin-X UF concentrators Alkaline Phosphatase Colorimetric Assay kit

Alkaline phosphatase Assay Colorimetric Embryonic Fugene 6 Human Kidney Promega Protein Protein a Sema4d Semaphorin

Corresponding Organization :

Other organizations : The Francis Crick Institute, King's College London, Icahn School of Medicine at Mount Sinai

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Variable analysis

independent variables

Transfection of HEK293T cells with Sema4D-AP in pcDNA3 vector using Fugene 6 reagent

dependent variables

Secretion of recombinant Semaphorin 4D protein fused with AP
Protein concentration of recombinant Semaphorin 4D-AP

control variables

Manufacturer's protocol for Fugene 6 reagent
Spin-X UF concentrators with 100kDa cutoff (Millipore)
Alkaline Phosphatase Colorimetric Assay kit (Abcam)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

Annotations

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