I transfected low-passage healthy Sf9 cells with PCR-verified bacmid(Bac-to-Bac system) and saw all the signs of infections during transfection and baculovirus amplication of P1 and P2,but I could not see the specific band with Western blotting and SDS-PAGE for P2 and P3 and P3 virus-infected(with varying MOI and time) cell lysates(cells were harvested when 70% was dead,as suggested by the manual).Thanks.The Bac-to-Bac manual says"Viral stock contains a mixture of recombinant and non-recombinant baculovirus",however,the PCR of purified Bacmid gives only a specific band of expected size,without a 300bp band.Can anyone give me some suggestion?
Usually, protein expressed in sf9 couldn't be detectecd by SDS-PAGE. Since the level is raletivly low. What's antibody you used for western-blot detection? does the protein designed intracellular or extracellular expression? how do you make sample for loading on western-blot?
Dear Dr. Wang,
Thanks very much for your kind reply.
Sigma M2 Flag was used to detect the Flag-taged protein in western blot.The protein is designed for intracellular expression,as shown by many previous studies expressing the same proteins with the same Bac-to-Bac system.The infected cells were harvested and lysed in 50mMTris-Cl pH7.5, 500mM NaCl,sonicated,centrifuged and boiled before SDS-PAGE or WB.The SDS-PAGE was used as suggested by the Bac-to-Bac manual and other studies(http://www.scielo.br/img/fbpe/bjmbr/v30n8/html/2685fig2.htm).
The attached is the PCR result of the Bacmid,bacmid A,C and D were used for protein expression.As shown in the figure,bacmid B contains a mixture of recombinant and non-recombinant one,is it sure that the Bacmid A,C,D and E contains only recombinant ones?
Can you give me some suggestion?
Thanks.
Best,
Xiaoqiang
Dear Dr.Khan,
Thanks very much for your kind reply.
Cells were harvested when 70% was dead when amplifying the P1 and P2 baculovirus.To test the protein expression,P3-infected cells were harvested 3-4 days post-infection with varying MOI.I did not determine the titer of the virus,however,64ml cells(at the density of 2 × 106 cells/mL) were infected with 0.3ml P2 baculovirus,and P3 virus was collected 5 days post-infection when 60-70% cells were dead and shown obvious signs of infection,so I guess the titer of the P3 virus will be high.Can we use the SDS-PAGE to detect the protein expression of the P3-infected cells?
Besides,on the basis of the PCR results shown above,is it sure that the Bacmid A,C,D and E contains only recombinant ones?
I will follow you advice next time and skipping the lysis.
Thanks.
Best,
Xiaoqiang
If you are using a anti-FLAG antibody, you can try to get hold of another FLAG tagged protein to use as a control. A couple of lanes with varying concentrations of the control protein can give you an idea of your sensitivity to detect, as well as function as verification that the transfer from gel to membrane is working.
Hi Xiaoqiang,
I recommend that you titre your virus. It will help indicate or eliminate low titre as a cause to your problem. Signs of infection and cell death are not by themselves an indication of high titres. It also lets you know that your MOI of your expression is near optimal.
It is also a good idea to test different harvest times for you expression, 48, 72 & 96 hours are the usual ones.
-Richard
p.s. your PCR on the bacmid looks fine, I'd not worry about them.
Dear Tang Xiaoqiang,
If you are looking at whole protein in the baculovirus cells, I would recommend you try different protein precipitation methods like TCA-Acetone etc., (refer any good article on 2-D electrophoresis of hemocytes/ hemolymph of insects )
Dear all,
Thanks for all your valuable suggestion,all advice will be tested in my late experiment.
Best,
Xiaoqiang
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