Olga makes some good points. But also remember that virus stocks don't last forever. If you are using the same original stock and it is >6 months old, you may get reduced expression compared to previously. You might want to go back to the plasmid and regenerate your P1. We find this can usually restore levels.

Hi guys,

thanks for the feedback. I have actually tested different MOIs and timepoints before on the parent protein and the mutant protein. I found a higher MOI around 10 works well but for the parent protein but MOIs of ~30 seems to do the trick for the mutant, which only differs from the parent by having an 20 a.a. internal deletion. My parent protein expresses very well and is secreted into the media whereas this mutant protein has low expression and remains in the cells. The reason I ask about viability is because I did my optimization experiments for the mutant then used the supposed optimal conditions for a larger scale expression and I got no protein. The only difference between the 2 experiments was that the viability this time was much higher (90%) than when it did work (60%). Also my current viral stocks are only a couple months old but they were made by infecting cells with older viral stocks so maybe I reamplify my virus. However, we do use an in house transfer vector which transfects by homologous recombination

Thanks again

All the points made above are valid. I agree that viral stock will be good only for few months at 4C. But I have used until 6 months without problem from the V1 stock( always kept at 4c and wrapped in aluminium foil). My personal experiene is with MultiBac and in this case we usually depend on the YFP expression level from the viral genome to help us get a rough idea when to harvest and the MOI.

In my experience most of the proteins are well expressed or maximally expressed 72-96hours after cells stop dividing in the culture suspension. But there are always exceptions hence its always a good idea to use the small volume of inoculation (1ml to 3ml of V1 in your case since you use homologous recombination method and also since scale up didnt seem to work ) to a 50ml ml culture and determine the expression by SDS PAGE until 96 or 120hours after cells stop dividing. you only have to take 1 million cells each time and do SDS PAGE. Its never a good idea to use inoculation volumes that stop the cell division immediately after V1 addition. GOod luck. Hope this helped

thanks for the advice. I think I need to do another optimisation and recheck the titre.