pFBDM-PS1(wt)-γ-secretase

Protocol tips

Upstream tips	Protocol tips	Downstream tips
	The recombinant or control (empty) EmBacY baculoviral DNAs were transfected into adherent cultures of Sf9 cells (1 × 106 cells) using FuGENE®HD Transfection Reagent (Promega), using the manufacturer’s protocol. Initiation of YFP expression was taken as an indication of recombinant protein expression. The cells were monitored every 24 hours by bright-field microscopy to observe cell membrane disruption, indicating initial stages of viral mediated cell lysis.

Protocol tips

The recombinant or control (empty) EmBacY baculoviral DNAs were transfected into adherent cultures of Sf9 cells (1 × 106 cells) using FuGENE®HD Transfection Reagent (Promega), using the manufacturer’s protocol. Initiation of YFP expression was taken as an indication of recombinant protein expression. The cells were monitored every 24 hours by bright-field microscopy to observe cell membrane disruption, indicating initial stages of viral mediated cell lysis.

Publication protocol

The recombinant or control (empty) EmBacY baculoviral DNAs were transfected into adherent cultures of Sf9 cells (1 × 106 cells) using FuGENE®HD Transfection Reagent (Promega), using the manufacturer’s protocol. Initiation of YFP expression was taken as an indication of recombinant protein expression. The cells were monitored every 24 hours by bright-field microscopy to observe cell membrane disruption, indicating initial stages of viral mediated cell lysis. Conditioned media containing initial baculoviral particles (V0) was collected at <50% cell viability and used to infect fresh 10 ml Sf9 (1 × 106 cells/ml) suspension culture to generate V1. The V1 baculovirus was used to infect 50 ml of Sf9 culture to generate baculovirus V2. The baculovirus V2 was saved in 5 ml aliquots at 4 °C for short term or −80 °C long term and used to infect larger Sf9 cultures. The V2 viral titre was determined using the baculoQUANT titration kit (Oxford Expression Technologies, UK), in accordance with the manufacturer’s protocol. Briefly, recombinant baculovirus DNA was extracted from viral particles and baculoviral gene gp64 was measured using qPCR and viral titre was estimated by plotting against a standard curve. Recombinant baculoviruses were pelleted from 80 µl of conditioned media by centrifuging at 16,000 × g for 5 minutes at room temperature. Next, the recombinant and internal standard baculoviruses (supplied) were resuspended in 20 µl of lysis buffer (supplied) and lysed in a thermocycler and heating to 65 °C for 15 minutes, 96 °C for 2 minutes, 65 °C for 4 minutes, 96 °C for 1 minutes, 65 °C for 1 minutes, 96 °C for 30 seconds. Next, 2 µl of recombinant baculovirus DNA was mixed with qPCR reagent (12.5 µl) and baculoviral gp64 gene primers and probes (3 µl) (supplied) to a final volume of 25 µl. The qPCR reactions were carried out in triplicates for each sample in a ViiA7 Real-Time PCR System (Applied Biosystems). Mean Ct values of unknown samples were plotted against the standard curve to obtain plaque forming unit (PFU)/ml of conditioned media.

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Protein Expression Eukaryotic cells - S. frugiperda γ-secretase using pFBDM-PS1(wt)-γ-secretase from Giuseppe Verdile, School of Biomedical Sciences, Faculty of Heal.

Manufacturer protocol

Download the product protocol from Giuseppe Verdile, School of Biomedical Sciences, Faculty of Heal for pFBDM-PS1(wt)-γ-secretase below.

Videos

Check out videos that might be relevant for performing Protein Expression Eukaryotic cells - S. frugiperda γ-secretase using pFBDM-PS1(wt)-γ-secretase from Giuseppe Verdile, School of Biomedical Sciences, Faculty of Heal. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.