I am purifying my secreted protein expressed in insect cells with a His-tagged purification column and getting no yield of my protein. Why and what can I do?

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Answer

Media used to culture insect cells usually have an acidic pH (6.0-6.5) or contain electron-donating groups that can prevent binding of the 6xHis-tagged protein to Ni-NTA. Amino acids such as glutamine, glycine, or histidine are present at significantly higher concentrations in media for growing insect cells than in media for growing mammalian cells, and compete with the 6xHis-tagged protein for binding sites on Ni-NTA matrices. Grace’s medium (Life Technologies), for example, contains approximately 10 mM glutamine, 10 mM glycine, and 15 mM histidine.

Dialysis of the medium against a buffer with the appropriate composition and pH (8.0) similar to the lysis buffer recommended for purification under native conditions usually restores optimal binding conditions. Note that depending on the medium used, a white precipitate (probably made up of insoluble salts) can occur, but normally the 6xHis-tagged protein remains in solution. This can be tested by either protein quantitation if using a protein-free medium or by monitoring the amount of 6xHis-tagged protein by western-blot analysis. After centrifugation, 6xHis-tagged protein can be directly purified from the cleared supernatant.

Answer Id: E9456

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My transduced cells are floating up and look dead, but the Cellfectin™ II Reagent alone control plate is fine. What could be happening?

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Answer

This may be due to contamination or cytotoxicity from the bacmid prep. Make sure to include a negative control that is the bacmid only without Cellfectin™ II Reagent. Additionally, use the PureLink™ HiPure plasmid prep kit, not the silica-based miniprep kit for bacmid prep.

Answer Id: E9447

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I’m getting a low-titer P1 viral stock and would like to generate a high-titer stock. What should I do?

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Answer

To get a high-titer stock, reinfect cells with the P1 stock and generate a P2 high-titer stock. Follow the directions in the BaculoDirect™ manual on page 18 to generate your P2 stock.

Answer Id: E9460

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You offer competent cells in Subcloning Efficiency™, Library Efficiency™ and MAX Efficiency™. How do these differ?

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Answer

There are a few exceptions, but in general the difference is in guaranteed transformation efficiency as follows:
Subcloning Efficiency™ cells are guaranteed to produce at least 1.0 x 10E6 transformants per µg of transformed pUC19 or pUC18 supercoiled plasmid
Library Efficiency™ cells are guaranteed to produce at least 1.0 x 10E8 transformants per µg pUC19 or pUC18 DNA
MAX Efficiency™ cells are guaranteed to produce at least 1.0 x 10E9 transformants per µg pUC19 or pUC18 DNA

Answer Id: E3869

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I don’t see any signs of cell infection 72 hours after infection with my P2 viral stock. Why is this?

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Answer

Check the MOI. It may be low because the titer of the P1 virus is lower than what was estimated.

Answer Id: E9450

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Are there preferred secretion signals for use with baculovirus?

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Answer

Insect-derived signal peptides and/or prosequences cannot always enhance the expression and/or secretion of foreign secretory pathway proteins in the baculovirus system. Please see the following references:

- Jarvis DL, Summers MD, Garcia A Jr, Bohlmeyer DA (1993) Influence of different signal peptides and prosequences on expression and secretion of human tissue plasminogen activator in the baculovirus system. J Biol Chem 268(22):16754-16762.
- Tessier DC, Thomas DY, Khouri HE, Laliberte F, Vernet T (1991) Secretion of a plant protein in the baculovirus system was enhanced when its signal peptide was replaced with an insect-derived signal peptide. Gene (Amst.) 98:177-183.

Answer Id: E9410

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My PCR analysis of the recombinant bacmid gives no product. What could be the cause of this and do you have any recommendations on how to fix this?

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Answer

Please see the possible causes and suggestions we have to alleviate this problem:

- Use pure proofreading polymerase for analysis: Use a Taq-based polymerase for the analysis.
- Insert is very long and causes difficulties in PCR: Instead of using both M13 forward and reverse primers, use one gene-specific primer paired with the M13 primer of your choice.
- Long GC-rich stretches in the gene of interest: Consider using DMSO (up to 8%) in the PCR reaction.

Answer Id: E9445

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I see a precipitate in my ganciclovir solution. What can I do?

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Answer

Warm the ganciclovir solution in a water bath at 37 degrees C for 5-10 min, then vortex for a few minutes. The precipitate should go back into solution.

Answer Id: E9458

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Both transfected cells and Cellfectin™ II Reagent alone negative control cells are dead within 36 hours. What could cause this?

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Answer

There are several possibilities:

- Using media containing antibiotics during transfections.
- Plating cells at too low a density: We recommend at least 70% confluence.
- Using cells at too early a passage: We recommend growing cells for at least 5 passages before using them for transfection.
- Contamination because of no pen/strep after the transfection: After 5-8 hr incubation with the transfection mixture, remove the mixture and add antibiotics containing media/well.

Answer Id: E9448

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I cannot detect any recombinant fusion protein after using the BaculoDirect™ Expression Kit. What could be the cause for this and what do you suggest I try?

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Answer

Please check the construction of your entry clone, and ensure that the insert is in frame with the vector. Analyze the recombinant viral DNA by PCR to confirm the correct size and orientation of your insert after the LR reaction. Sequence your PCR product to verify the proper reading frame for expression of the epitope tag.

Answer Id: E9461

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What is the difference between the Bac-N-Blue™ expression system and the Bac-to-Bac™ expression system?

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Answer

In the Bac-N-Blue™ system, recombination between the transfer vector and the baculovirus DNA occurs in insect cells. The Bac-N-Blue™ vector is a linearized AcMNPV derivative that contains an incomplete (3’) lacZ fragment. The corresponding transfer vector contains a 5’ lacZ fragment. Upon homologous recombination, the recombinant Bac-N-Blue™ baculovirus DNA will have a complete lacZ gene that is under the control of the PETL promoter. Thus, recombinant Bac-N-Blue™ baculovirus will provide blue plaques in the plaque assay and can be easily identified. In the Bac-to-Bac™ expression system, recombination or site-specific transposition between transfer and baculovirus DNA occurs in E. coli (DH10Bac). In the Bac-to-Bac™ expression system, selection of colonies containing recombinant baculovirus DNA occurs in the presence of Luria Agar plates with 50 ™μg/mL kanamycin (bacmid), 7 ™μg/mL gentamycin (pFastBac™), 10 ™μg/mL tetracycline (helper plasmid), 100 ™μg/mL Bluo-gal, and 40 ™μg/mL IPTG.

Answer Id: E9419

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Is there a packaging limit for the baculovirus?

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Answer

The baculovirus rod will continue to elongate as required to package the DNA. Thus, the system could theoretically accommodate hundreds of Kb. Standard cloning techniques will limit the insert size before packaging limits become an issue.

Answer Id: E9411

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I'm trying to generate recombinant bacmid DNA using the Bac-to-Bac™ expression system and all of my colonies are blue. What could be the cause of this and what should I do?

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Answer

Please review the following possibilities and recommendations:

- pFastBac™ DNA used for transformation was of poor quality: Use purified plasmid DNA for transformation and check the quality of your plasmid DNA.
- Gentamicin omitted from plates: Prepare fresh selective plates containing 50 μg/mL kanamycin, 7 μg/mL gentamicin, 10 μg/mL tetracycline, 100 μg/mL Bluo-gal, and 40 μg/mL IPTG.

Answer Id: E9439

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When should DMSO, formamide, glycerol and other cosolvents be used in PCR?

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Answer

Cosolvents may be used when there is a failure of amplification, either because the template contains stable hairpin-loops or the region of amplification is GC-rich. Keep in mind that all of these cosolvents have the effect of lowering enzyme activity, which will decrease amplification yield. For more information see P Landre et al (1995). The use of co-solvents to enhance amplification by the polymerase chain reaction. In: PCR Strategies, edited by MA Innis, DH Gelfand, JJ Sninsky. Academic Press, San Diego, CA, pp. 3-16.

Additionally, when amplifying very long PCR fragments (greater than 5 kb) the use of cosolvents is often recommended to help compensate for the increased melting temperature of these fragments.

Answer Id: E1320

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My viral stock used to work well from my Bac-to-Bac™ baculovirus expression system, but after a couple of months, there is much less protein expression. Why is this?

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Answer

Please review the following possibilities and solutions:

- Viral stock was amplified using high MOI originally: Go back to the lower-passage viral stock and do a low-MOI amplification.
- Did not spin down and get rid of cells when harvesting viral supernatant: Go back to the lower-passage viral stock and do a low-MOI amplification; if this viral stock is P2, this stock can be used in amplification.
- For some genes, the virus can become very unstable: Free the aliquoted P2 viral stock and do one run of amplification after reviving.

Answer Id: E9455

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