Is it normal to observe a precipitate in the ganciclovir solution after thawing?

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Answer

A precipitate may sometimes appear after thawing the ganciclovir solution. If this occurs, heat the solution to 37C in a water bath for 5-10 minutes and vortex a few times. The precipitate should go back into solution. For future use, it is best to aliquot the remainder and store at -20C.

Answer Id: E4499

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4395bad605f15f77436392c3d1009767_FAQ

Is baculovirus good for expressing toxic proteins?

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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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684b1dcde50ca096380622a39238ff89_FAQ

When should I harvest my protein after I have inoculated my insect cells with recombinant virus?

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Answer

Peak expression of protein in insect cells is dependent on the multiplicity of infection (MOI), expression time, and the protein being expressed. Guidelines to optimize your system include using an MOI of 5-10 and an expression time of 48-72 hours. Protein expressed at times later than 72 hours may be processed aberrantly, because the large virus load can cause a breakdown of cellular processes.

Answer Id: E9396

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2241e959dd3caaf1b079940794f780d2_FAQ

What are some general suggestions for transfection in baculovirus expression systems?

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Answer

Please follow the recommendations below:

- Cells should be in excellent health, of their low passages (5-15), in log-phase growth, with viability >95%
- DNA must be of high purity, free of endotoxin
- No antibiotics should be used during transfection
- Cellfectin® reagent has to be completely resuspended
- Include controls (media control, DNA control, and transfection reagent control) for comparison and troubleshooting

Answer Id: E9397

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c12faac49f4675b35200e164aa9f06a2_FAQ

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

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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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e22d16827b1fff4b1a21cb4d8a5a79c8_FAQ

What does viral infection look like in early, late, and very late stages?

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Answer

Please see the description below of the different stages of viral infection:

Early
- Increased cell diameter-a 25-50% increase in the diameter of the cells may be observed.
- Increased size of cell nuclei-the nuclei may appear to "fill" the cells.

Late
- Cessation of cell growth-cells appear to stop growing when compared to a cell-only control.
- Granular appearance
- Signs of viral budding-vesicular appearance of cells.
- Viral occlusions-few cells will contain occlusion bodies, which appear as refractive crystals in the nucleus of the insect cell.
- Detachment-cells release from the dish or flask.

Very late
- Cell lysis-a few cells may fill with occluded virus, die, and burst, leaving signs of clearing in the monolayer.

Answer Id: E9400

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e74a57a71b0d2141bef6df29a912c244_FAQ

Do I need to purify my recombinant virus away from an uncut or non-recombinant viral DNA?

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Answer

Yes. Contamination of your recombinant DNA with uncut (occ+) DNA will lead to dilution of your recombinant virus over time because, in general, uncut (wild-type, occ+) virus infects and replicates at higher efficiency than recombinant virus. Also, initiating expression studies with a pure, single virus population will ensure reproducible results.

Answer Id: E9401

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7f725f23cb1485ca7b845a5537bc580c_FAQ

How can I store my viral stock?

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Answer

If the medium is serum-free, add serum to 10%. Serum proteins act as substrates for proteases and therefore prevent degradation of viral coat proteins. Store viral stocks at 4 degrees C, and protect from light. Aliquots can be stored at -80 degrees C, but viral titer should be checked before use, as freeze/thaw cycles of the virus can result in a 10- to 100-fold decrease in viral titer.

Answer Id: E9402

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f75b0664a214df01887ebf3d7fc5d526_FAQ

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

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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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c861947005d53eb58e8c07081ddcb65f_FAQ

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

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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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2dbb2ff014f307e616e5eb8b0ac1e746_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?

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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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4fb8d04518b64693eedb9e2274320bc5_FAQ

Is it necessary to include a Kozak sequence for expression of recombinant proteins in insect cells?

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Answer

While the importance of a Kozak consensus sequence in translation initiation has been demonstrated in mammalian cells, there seems to be some debate as to whether the Kozak rules are as stringent in insect cells. The only way to determine its importance would be a direct comparison of expression of the same protein from different initiation sequences. Even then, the rules for optimal expression of one protein may not hold for another. Here are two references which indicate that a Kozak consensus sequence does not have any effect on efficiency of expression in insect cells:

- Hills D, Crane-Robinson C (1995) Baculovirus expression of human basic fibroblast growth factor from a synthetic gene: role of the Kozak consensus and comparison with bacterial expression.
- Biochim Biophys Acta 1260(1):14-20.
- Ranjan A, Hasnain SE (1995) Influence of codon usage and translational initiation codon context in the AcNPV-based expression system: computer analysis using homologous and heterologous genes. Virus Genes 9(2):149-153.

Answer Id: E9406

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1f55e7cff93875e1a9b3543fb06e4307_FAQ

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

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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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e196f61d6dbba2513e7d2ab6f475f441_FAQ

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

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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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4324c890dce0a0b478b0a61d154e1c04_FAQ

What is the thymidine kinase (TK) gene for?

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Answer

The TK gene is for negative selection of non-recombinant virus using ganciclovir.

Answer Id: E9417

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627985752772d176e696740a416455ad_FAQ