InsectDirect™ System

InsectDirect Home   •  How does use of the       InsectDirect System benefit me?   •  What does the InsectDirect System offer?      •  Insect Cell Expression Vectors      •  Insect GeneJuice® Transfection Reagent      •  Insect RoboPop™         Ni-NTA His•Bind® Purification Kit         •  A brief protocol for the RoboPop kit         •  Comparison of traditional baculovirus             method and InsectDirect System method         •  Small- and large-scale Sf9 suspension             culture transfections

Small-scale (10 ml) and large-scale (1 liter) Sf9 suspension culture transfections

Sf9 cells in 10-ml suspension cultures (1 × 10⁶ cells/ml) were transfected with 20 µg pIEx™ recombinant plasmid using Insect GeneJuice® Transfection Reagent (Figure A). Total culture extracts were prepared 48 h later by adding Insect PopCulture® Reagent, followed by the addition of Benzonase® Nuclease. Samples were removed at that point to assess total cell protein. Ni-NTA His•Bind® Resin was then added directly to the extracts. The samples were processed robotically using a MultiPROBE® II HT EX Liquid Handling Station (PerkinElmer). Samples of the crude and purified fractions from each transfection were analyzed by SDS-page (10-20% gradient). Purified protein yields were determined by BCA assay.

		Lane Sample (Yield)  1.  Perfect Protein™ Markers,       15–150 kDa  2.  Heat Shock Protein 1,       Total cell protein,       supercoiled plasmid  3.  Heat Shock Protein 1, Eluate,       supercoiled plasmid (483 µg)  4.  Heat Shock Protein 1,       Total cell protein,       linearized plasmid  5.  Heat Shock Protein 1, Eluate,       linearized plasmid  6.  Heat Shock Protein 2,       Total cell protein  7.  Heat Shock Protein 2, Eluate (314 µg)  8.  Perfect Protein Markers,       15–150 kDa  9.  Phospholipase, Total cell protein 10. Phospholipase, Eluate (213 µg)
	A. Heat shock proteins and phospholipase, 10-ml scale
Four 250-ml suspension cultures were seeded with Sf9 cells (1 × 106 cells/ml) in serum-free BacVector® Insect Cell Medium (Figure B). A pIEx/protein kinase isoform construct was added to the diluted Insect GeneJuice Transfection Reagent, incubated for 15 min at room temperature, and then added to the flasks. After 48 h incubation at 28°C, Insect PopCulture Reagent and Benzonase Nuclease were added directly to each flask and incubated for 15 min at room temperature to lyse the cells. The His•Tag® fusion protein was manually purified on Ni-NTA His•Bind Resin according to the standard protocol. Protein yield was quantified by a modified Bradford assay.
		Lane Sample (Yield)  1.  Perfect Protein Markers, 15–150 kDa  2.  Protein Kinase, Total cell protein  3.  Protein Kinase, Eluate (13.3 µg)
	B. Protein kinase, 1-liter scale