Hi,
I am new with baculovirus expression system in insect cell (Sf9). I am trying to express a human protein (around 65 kDa, secreted) in Sf9 cells using Bac-to-Bac HBM TOPO Secreted Expression System from Invitrogen. I included pFastBac GUS as a positive control for checking the expression. I collected the medium at in interval of 24, 48, 72 and 96 h, and then assessed the B-glucuronidase expression with the chromogenic indicator X-Gluc (Invitrogen, #R0851). I mixed 5ul of 20 mg/ml X-Gluc (in DMSO) with 50 ul of cell-free medium, but I did not manage to see any color developed, as suggested in the protocol, even after couple of hours.
Could you please help me what might be the issue?
Best,
Dipankar
At first please check whether you have bacmid in cells. Did you try to prepare a cellular extract of your Sf9 cells ? and assay for in vitro GUS activity to see if there is or not GUS produced in your cells. Use p-Nitro-D-glucuronic acid as a substrate.
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