Resources

When to Passage Sf9 Cells

An important factor in baculovirus expression is assessing when to passage Sf9 cells, which are commonly used to make recombinant viruses or for protein production.  In our last blog we talked about how to make the transition from monolayer to suspension cultures and how maintain cells in a healthy state during this time. To define what is meant by ‘healthy’ cells,  Figure 1 shows a sample of healthy Sf9 cells

Suspension Sf9 Cells from Monolayer Cultures

The transition to suspension Sf9 cells from monolayer cultures is not difficult but does depend on harvesting the attached cells at the right time. Too early and it is extremely hard to detach the cells from the culture flask. Too late and the cells will be in such poor condition that they may not recover when in suspension. Trying to describe in words how the monolayer culture should look prior

Healthy Cells Over the Holiday

We want to talk about maintaining healthy cells over the holiday period spanning Christmas and New Year.  (Some of our regular readers may recognize most of the following from blogs we have posted in previous years, but we think it is worth repetition for new users of the baculovirus-insect cell system). Most labs, academic and industry, will have some time off over the next few weeks. It is often a

Reviving Cells from Liquid Nitrogen

The process of reviving cells from liquid nitrogen, or indeed from transport on dry ice, can be a little difficult on occasion.  Two events at OET recently have highlighted this. Firstly, we received a new cell line that came with a very complicated procedure for its culture – it is a mammalian species, quite different from your run of the mill HeLa’s and HEK293’s.  Its culture is known to be

Co-infection with Baculovirus Vectors for Multiple Protein Expression

A question that comes up from time to time is the use of co-infection with baculovirus vectors for multiple protein expression in insect cells.  The need arises if you have produced a number of baculovirus vectors that each synthesise a component of a multi-subunit complex, such as an enzyme or a virus like particle.  Intuitively, you know that it is probably necessary to add many virus particles per cell to

Baculovirus Transfer Vectors Compatible with flashBAC™

A question we are asked from time to time is to identify baculovirus transfer vectors compatible with our flashBAC™ system. We obviously nominate our own extensive range that is available but frequently someone has an old plasmid from years ago that they want to reuse. Originally they may have paired it with a linear DNA system such as BaculoGOLD or BacPAK6 or even wild type DNA where recombinant virus selection

Variable Expression of Multi-Component Protein Complexes

Variable expression of multi-component protein complexes is increasingly necessary as more sophisticated projects are attempted.  But how can this be achieved?  Most of our expression work is based on the baculovirus system, in particular our flashBAC™ vectors that permit easy recombinant virus generation in a one-step process.  These insect-specific viruses offer an ideal platform for producing a wide range of recombinant proteins including nuclear, cytoplasmic, membrane-bound and secreted examples. But

Intermediate Scale Recombinant Protein Production

Between initial tests and larger scale up you may want to conduct intermediate scale recombinant protein production using baculovirus vectors.  If you are working with several constructs then this can begin to get expensive of cell culture vessels such as the ubiquitous 125ml conical flasks, in which you can amplify 25-30mls of virus-infected cells.  Space on shakers can also be at a premium in some labs. To address both the

Adapting Insect Cells to a New Growth Medium?

Have you ever had the experience of adapting insect cells to a new growth medium?  We recently had to do this for a client project because they had used a particular medium previously and were keen to maintain the same conditions to continue the work with us. This process made us realise that switching to a new serum-free insect cell culture medium can result in short term stress responses in

Baculovirus-Expressed Secreted Recombinant Proteins

A question that often comes up from clients is what is the best signal peptide to use for baculovirus-expressed secreted recombinant proteins?  While we have discussed the best flashBAC™ variant to use for the production of secreted proteins in a previous blog, we have never considered the role of the signal peptide. In fact, while writing this blog, we realised that there was much that we didn’t know, so will

Sequence Errors in Your Gene for Expression? (Parts I + II)

If you don’t achieve the levels of protein expression expected in your project, have you ever considered that there may be sequence errors in your gene?  This applies to all expression systems, not just those based on baculoviruses.  We should be clear that we are not talking about sequence errors in your gene after it has been synthesized by an out sourcing company.  Quality control by such companies is such

Quick Guide to Plaque Assays

Last week we ran a short training course on baculovirus expression vectors.  As part of this we put together a one page guide for carrying out plaque assays.  This might be useful to those of you looking for something you can have on the lab bench while conducting your own plaque assays.  We have added a image of one of our 12-well plate-based plaque assays below as an example.  The

Transduction of Whole Porcine Kidneys with BacMAMs

One of the unexpected offshoots of the baculovirus expression system has been its development as a vehicle for the delivery of genes to mammalian cells.  These vectors are otherwise known as BacMams.  Although baculoviruses are unable to replicate in mammalian cells, they can enter them via receptor-mediated endocytosis.  In Frederick Boyce’s original paper the Rous sarcoma virus promoter was used to express beta-galactosidase in the HepG2 human liver cell line. 

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