Dhananjay, Hopefully you are using the 'Bac to Bac' system, not the older version 'Bac-N-Blue'. I'd recommend getting a high-titer virus to use as a positive control for infection. Baculovirus is challenging because before you can check for protein expression, you need to do virology experiments. If you don't have a high-titer virus stock, you can't conclude anything about your expression level. Using a positive control virus will help you learn to recognize the signs of infection. Then you can judge if you problem is related to virus amplification, or expression optimization. If I had to make a wild guess, I would say you probably don't have a high-titer stock yet, as a result you don't see protein expression, and you conclude that it is 'not working'. Consider using the pIB vectors for transient expression alongside with baculovirus, the transient experiments are less complicated and offer a quicker readout. Good luck,
Nat PS: One useful trick: If you are struggling to boost the titer of a early virus stock (ie. P0, P1), take a t25 flask of Sf9 cells (50-70% confluent), remove the media, place 1 ml of your low-titer virus stock on the cells, rock for 1 hr, remove virus and replace with fresh media. Let that go for 3-5 days and hope you see signs of infection. If not you probably don't have any virus and need to repeat the transfection and/or miniprep. On Fri, Dec 6, 2013 at 6:32 PM, dhananjay kulkarni < [email protected]> wrote: > Dear all, > Does any one has protocols for expression and making stable virus for SF-9 > Cell, I was trying > with Invitrogen manual but somehow it is not working, any alternative > protocols which works > quickly? I am new to thins insect cell expression. > your suggestion and protocols are really helpful > Thanking in advance > > > > Dhananjay.K > Post Doctoral Research Associate > Department of Biochemistry > Indian Institute of Science > Bangalore > India > > <http://sigads.rediff.com/RealMedia/ads/click_nx.ads/www.rediffmail.com/signatureline.htm@Middle?> > Get your own *FREE* website, *FREE* domain & *FREE* mobile app with > Company email. > *Know More > >*<http://track.rediff.com/click?url=___http://businessemail.rediff.com/company-email-hosting-services?sc_cid=sign-1-10-13___&cmp=host&lnk=sign-1-10-13&nsrv1=host>
