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

A brief protocol for the RoboPop™ kit

Insect PopCulture Reagent is added directly to the cell culture, followed by the addition of Benzonase Nuclease. The lysate is transferred to an appropriately sized tube or deep-well plate. After Ni-NTA His•Bind® resin is added and mixed, the lysate-resin slurry is transferred to the Filter Plate, washed, and target proteins are eluted into the Collection Plate on a vacuum manifold.

See Overview of reagent requirements for various culture sizes for more information

		Transfected cells 10 ml insect suspension culture Incubate 48 h
		Lysate generation Insect PopCulture Reagent Benzonase Nuclease Mix 15 min at room temperature
		Binding His•Tag fusion proteins Add Ni-NTA His•Bind resin Transfer to tube Mix 30 min at 4°C
	filter plate	Purify bound proteins Transfer to filter plate on vacuum Capture resin, remove flow-through Wash Elute purified protein into collection plate
	collection plate
		Purified protein
Overview of reagent requirements for various culture sizes
Culture size Sf9 cells Plasmid DNA dilution in BacVector® Medium Insect GeneJuice Transfection Reagent dilution in BacVector Medium Insect PopCulture Reagent Benzonase Nuclease His•Bind Ni-NTA Resin Resin pool applied to column His•Bind Ni-NTA 1X Wash Buffer His•Bind Ni-NTA 1X Elute Buffer 10 ml 9 ml at 1.0 × 106/ml 20 µg + 0.5 ml 60 µl + 0.4 ml 0.5 ml 5 µl 0.25 ml 96-well filter plate 2 × 1.5 ml 0.5 ml 100 ml 90 ml at 1.1 × 106/ml 200 µg + 5 ml 1 ml + 4 ml 5 ml 50 µl 2.5 ml 30 mm × 20 cm 2 × 15 ml 7.5 ml 200 ml 180 ml at 1.1 × 106/ml 400 µg + 10 ml 2 ml + 8 ml 10 ml 100 µl 10 ml 30 mm × 20 cm 2 × 30 ml 15 ml 1 liter (4 × 250-ml cultures) 225 ml at 2.5 × 108/ml per flask 500 µg + 12 ml per flask 2.5 ml + 10 ml per flask 12.5 ml per flask 125 µl per flask 25 ml to pooled lysate 30 mm × 23 cm 2 × 100 ml 75 ml