How does the NativePure™ Affinity Purification System work?

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Answer

pcDNA™ vectors with “capTEV™” tags (BioEase™ in vivo biotinylation peptide + 2 TEV protease recognition sites + 6X His tag) allow expression of biotinylated recombinant fusion proteins in mammalian cells. The protein complexes are purified under native conditions using streptavidin agarose included in the kit.

Answer Id: E12910

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I am working with a mouse cell line and would like to express my gene at high levels using one of your vectors with the CMV promoter. Do you foresee any problems with this approach?

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The CMV promoter is known to be downregulated over time in mouse cell lines. Hence, we recommend using one of our non-CMV vectors, such as those with the EF1alpha or UbC promoter, for long-term expression in mouse cell lines.

Answer Id: E9152

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What are the specifications of the NativePure™ Columns?

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The NativePure™ Columns hold up to 2 mL of chromatography resin and contain a 10 mL reservoir for sample or buffer. The column specifications are listed below:

-Pore size of purification columns: 30-35 microns
-Recommended flow rate: 0.5 mL/min
-Maximum flow rate: 2 mL/min
-Maximum linear flow rate: 700 cm/h
-Column material: polypropylene
-pH stability (long term): pH 3-13
-pH stability (short term): pH 2-14

Answer Id: E12911

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I'm not getting any recombinant protein or protein complexes following elution when using the NativePure™ Affinity Purification System. What could be causing this?

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Answer

-The expression level of your protein may be too low; ensure that the protein of interest is in vivo biotinylated.
-AcTEV™ cleavage is not efficient or the enzyme is inactive; ensure that the AcTEV™ protease is stored at -20 degrees C or -80 degrees C to prevent any loss in activity. You can try increasing incubation time or amount of enzyme to optimize cleavage.
-Protein complex not recovered in the soluble fraction; perform western analysis to check that protein complexes are recovered in the post-nuclear supernatant. If more than 50% of the protein complexes are remaining in the pellet, solubilize the protein complexes using mild, non-ionic detergents, such as NP-40, Triton™ X-100 or equivalent.
-Not enough sample was loaded; increase the amount used.
-Adherent cells are difficult to harvest using mild conditions; if you need to use trypsin treatment, do so for a short time.
-Protein is degraded; perform all purification steps at 4 degrees C. Check to make sure that the BioEase™ tag is not cleaved during processing or purification, and include protease inhibitors during cell lysis.

Answer Id: E12971

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Can I purchase the 5X LR Clonase™ buffer or 5X BP Clonase™ buffer separately?

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We do not offer the 5X LR Clonase™ buffer and 5X BP Clonase™ buffer as standalone products. They are available as part of the enzyme kits.

Answer Id: E9855

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Which competent E. coli do you recommend using for propagation of my Gateway™-adapted mammalian Destination vector?

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We recommend using One Shot™ ccdB Survival™ 2 T1R Competent Cells, Cat. No. A10460. This strain is resistant to the toxic effects of the ccdB gene. Note: Do not use general E. coli cloning strains, including TOP10 or DH5alpha™, for propagation and maintenance, as these strains are sensitive to ccdB effects.

Answer Id: E9153

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Do you have a recommended single-step protocol for BP/LR recombination?

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Yes, we have come up with a single-step protocol for BP/LR Clonase™ reaction (http://www.thermofisher.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.

Answer Id: E9857

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I'm recovering recombinant protein after NativePure™ affinity purification, but it is not complexed. What could be the cause of this?

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Complexes can dissociate during lysis preparation or purification. To minimize this, perform cell lysis using freeze-thaw cycles. Avoid trypsinizing cells or scraping cells. Perform cell lysis in the absence of NP-40, as some protein complexes may be unstable in its presence. Avoid vortexing the lysate during preparation, and perform all purification steps at 4 degrees C using chilled buffers

Answer Id: E12972

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I used a mammalian expression vector but do not get any expression of my protein. Can you help me troubleshoot?

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Answer

Here are possible causes and solutions:

- Try the control expression that is included in the kit
Possible detection problem:

- Detection of expressed protein may not be possible in a transient transfection, since the transfection efficiency may be too low for detection by methods that assess the entire transfected population. We recommend optimizing the transfection efficiency, doing stable selection, or using methods that permit examination of individual cells. You can also increase the level of expression by changing the promoter or cell type.
- Expression within the cell may be too low for the chosen detection method. We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot. Protein might be degraded or truncated: Check on a Northern. Possible time-course issue: Since the expression of a protein over time will depend upon the nature of the protein, we always recommend doing a time course for expression. A pilot time-course assay will help to determine the optimal window for expression. Possible cloning issues: Verify clones by restriction digestion and/or sequencing.

Answer Id: E9182

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I'm seeing additional or nonspecific proteins in the elute along with my protein of interest when using the NativePure™ Affinity Purification System. What can I do to eliminate this?

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Answer

Try including 0.1% NP-40 during the binding step to eliminate nonspecific binding. Include protease inhibitors during cell lysis to minimize protein degradation.

Answer Id: E12974

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Can I use E. coli containing an F' plasmid, such as Top 10F', to propagate a vector with the ccdB gene?

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Answer

While the F' plasmid does contain the ccdA gene that can inhibit or reduce the toxicity of the ccdB gene product, the ccdA expression level is likely to be too low, or inhibition may not be complete, and the bacteria would still be exposed to the ccdB gene product and thus not grow. Therefore, bacterial strains containing the F' plasmid are not recommended as hosts for propagation of ccdB containing vectors.

For propagation of Gateway™ vectors containing ccdB, we recommend the One Shot™ ccdB Survival™ 2 T1R Competent Cells (A10460), which were specifically designed for that purpose. However, please note that these cells are not validated for propagation of other ccdB-containing vectors like the older pZErO™ plasmids, and in most cases they are not expected to work due to very high levels of ccdB protein expressed in those vectors.

Answer Id: E3870

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Do you offer Gateway™ vectors for expression in plants?

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Answer

We do not offer any Gateway™ vectors for expression in plants.

Answer Id: E9854

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How can I move my gene of interest from a Gateway™-adapted expression clone to a new Destination vector as I have lost the entry clone?

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Answer

We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.

Answer Id: E9856

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I am using a mammalian expression vector that has the neomycin resistance gene. Can I use neomycin for stable selection in mammalian cells?

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Answer

No; neomycin is toxic to mammalian cells. We recommend using Geneticin™ (a.k.a. G418 Sulfate), as it is a less toxic and very effective alternative for selection in mammalian cells.

Answer Id: E9181

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Do you offer a GFP-expressing mammalian expression vector that I can use as a control to monitor my transfection and expression?

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Answer

We offer pJTI™ R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.

Answer Id: E9154

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