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Product FAQ

Is there a way to make baculovirus plaques more visible or distinctive?

Answer

You can stain the monolayer with neutral red or MTT to make the plaques more visible. Alternatively, you can allow the plates to develop for a few days longer (2-5 days on average) at room temperature to increase the contrast in recombinant plaques. However, the plaques stained with neutral red cannot be used for plaque purification and viral amplification.

Answer Id: E9427

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Product FAQ

Can I scale-up the production of recombinant protein using the baculovirus expression system? If so, what methods are available?

Answer

Yes, large-scale expression experiments can be performed. Please see below for different large-scale methods, requirements, added benefits, and references:
- Stirred bioreactor
- Airlift fermentor
- Insect larvae

Answer Id: E9403

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Product FAQ

To propagate more recombinant virus stock, what MOI should I use and when should I harvest the virus?

Answer

When propagating virus stock, use a low MOI (0.03-0.1) in order to avoid effects of defective interfering particles (DIPs). A low MOI, which ensures no more than 1 virion per cell, prevents the amplification of DIPs. A harvest time based on 15% cell viability is appropriate. NOTE: DIPs are nearly normal virus capsids containing genomes that are defective and are unable to undergo successful replication. While this "particle" is not infectious by itself, it can replicate when co-infected with normal virion, or with some other types of DI particles.

Answer Id: E9428

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Product FAQ

Could you please provide me with a protocol overview for the BaculoDirect™ system?

Answer

Begin your BaculoDirect™ experiments by cloning in your gene of interest into your Gateway™ Entry Vector, followed by the LR Clonase™ reaction into the BaculoDirect™ vector. Transfect this vector into your cells and grow for 3 days. On the 4th day, collect P1 viral stock. Re-infect cells, and grow for 3 days. On the 7th day, collect P2 viral stock. Infect cells, followed by harvesting of protein and purification on the 10th day.

Answer Id: E9415

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Product FAQ

What is the multiplicity of infection, and how can I calculate it?

Answer

The MOI, or multiplicity of infection, is the average number of viral particles that infect a single cell in a specific experiment. You can calculate the MOI with the following equation:
MOI (pfu/cell) = [titer (pfu) x viral stock volume (mL) used in inocula] / [cell density (cells/mL) x culture volume (mL)]

Answer Id: E9404

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Product FAQ

I’ve infected my cells and see large polyhedra in one cell and smaller polyhedra (more numerous) in a neighboring cell. Is this normal?

Answer

Yes, cells are infected with wild-type virus individually and will develop polyhedra at different rates until all the cells in the flask are infected. The polyhedra in cells will form in approximately 3-4 days, differing in size and number until they reach their maximum capacity and burst the cell, releasing tiny particles of virus into the medium.

Answer Id: E9473

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Product FAQ

After infecting T25 or T75 flasks with virus, when do you begin to see cell lysis and what percentage of cells will be lysed at certain time points?

Answer

This is dependent on how much virus is added. If cells are infected at an MOI of 5, usually cells are infected at 24 hours, and cells begin to lyse at around 65 hours. If less virus is used, this takes longer, and more virus takes less time.

Answer Id: E9429

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Product FAQ

What cell lines do you recommend using with the BaculoDirect™ system?

Answer

We recommend using Sf9 or Sf21 cells to generate high-titer viral stocks. We do not recommend using High Five™ cells to generate viral stocks due to lower transfection efficiency. Once you have generated your high-titer viral stocks, you can use Sf9, Sf21, High Five™, or Mimic™ Sf9 cells for protein expression.

Answer Id: E9416

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Product FAQ

Is it possible to co-infect insect cells with two different recombinant viruses in order to co-express, for example, two subunits of a protein?

Answer

Yes, it is possible. Several five-subunit proteins, such as human replication factor C, have been expressed using recombinant baculovirus. We recommend that a separate high-titer stock (HTS) of each subunit be produced to optimally express the multi-subunit protein. This way, the amount of each subunit expressed can be controlled by varying the multiplicity of infection (MOI) of each subunit's HTS. Please refer to the following articles for more information:

- Chen W and Bhal OP (1991) Recombinant carbohydrate and selnomethionyl variants of human choriogonadotropin. J Biol Chem 266(13):8192-8197.
- Chen WY and Bhal OP (1991) Selenomethionyl analog of recombinant human choriogonadotropin. J Biol Chem 266(15):9355-9358.
- Fabian JR, Kimball SR, Jefferson LS (1998) Reconstitution and purification of eukaryotic initiation factor 2B (eIF2B) expressed in Sf21 insect cells. Protein Expr Purif 13(1):16-22.

Answer Id: E9405

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Product FAQ

I’m getting mostly white/wild-type plaques instead of blue/recombinant plaques. What am I doing wrong?

Answer

This is typically an indication of poor homologous recombination. Check the plasmid/linear DNA ratio you used. If there are some blue plaques, however, expand those viruses and check for their protein. In our experience, they are correct, even if they were in relatively low abundance.

Answer Id: E9474

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Product FAQ

Is baculovirus good for expressing toxic proteins?

Answer

Yes, baculovirus is a good candidate for the problem of expressing toxic proteins (i.e., membrane proteins). The polyhedron promoter does not express at maximal levels until 18-24 hr after infection. The polyhedron promoter is active late in the lytic cycle. That being said, it is minimally active as early as 8 hours, so if the gene is very toxic, there may be a problem. The solution in that case would be to switch to an inducible expression system. Transmembrane proteins can often be difficult to express in any system.

Answer Id: E9395

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Product FAQ

What should I look for to indicate a successful transfection in which baculovirus is produced?

Answer

Adherent Sf9 cells round up and show a smaller contact point. Infected Sf9 cells in suspension culture round up and look larger when infected.

Answer Id: E9398

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Product FAQ

What is the optimal time for harvesting high-titer virus following infection? What happens if I let the cells go longer?

Answer

We recommend harvesting high-titer virus when there is 90% cell lysis. This takes approximately 5-7 days. If the cells go longer, the proteases released from the lysed cells will start to degrade viral surface proteins and result in less infectious virus.

Answer Id: E9430

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Product FAQ

I think I’ve added too much virus. How important is the MOI? What happens if too much virus is used?

Answer

An MOI of 5-10 is typically used. If too much virus is added, unfortunately the cells die too soon and the protein expression level goes down.

Answer Id: E9466

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Product FAQ

How do I determine the titer of my viral stock?

Answer

We recommend you perform a plaque assay to determine the titer of your viral stock. You may also perform a plaque assay to purify a single viral clone, if desired.

Answer Id: E9423

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