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BN3002

Product FAQ

What are the specifications of the NativePure™ Columns?

Answer

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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Product FAQ

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

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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Product FAQ

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

Answer

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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Product FAQ

Is it okay if my construct has an ATG that is upstream of the ATG in my gene of interest? Will it interfere with translation of my gene?

Answer

Translation initiation will occur at the first ATG encountered by the ribosome, although in the absence of a Kozak sequence, initiation will be relatively weak. Any insert downstream would express a fusion protein if it is in frame with this initial ATG, but levels of expressed protein are predicted to be low if there is a non-Kozak consensus sequence. If the vector contains a non-Kozak consensus ATG, we recommend that you clone your gene upstream of that ATG and include a Kozak sequence for optimal expression.

Answer Id: E9180

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Product FAQ

Do you offer Gateway™ vectors for expression in plants?

Answer

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

Answer Id: E9854

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Product FAQ

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

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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Product FAQ

I am using the NativePure™ Affinity Purification System. Some of the recombinant protein is in the flow through and wash fractions. What should I do?

Answer

This typically occurs when protein is overloaded. Load less protein or use more resin for purification.

Answer Id: E12973

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Product FAQ

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?

Answer

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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Product FAQ

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?

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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Product FAQ

How does the NativePure™ Affinity Purification System work?

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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Product FAQ

Are there common restriction sites that can be used to excise a gene out of a Gateway™ plasmid?

Answer

The core region of the att sites contains the recognition sequence for the restriction enzyme BsrGI. Provided there are no BsrGI sites in the insert, this enzyme can be used to excise the full gene from most Gateway™ plasmids. The BsrGI recognition site is 5'-TGTACA and is found in both att sites flanking the insertion site.
If a different restriction site is desired, the appropriate sequence should be incorporated into your insert by PCR.

Answer Id: E3317

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Product FAQ

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

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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Product FAQ

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

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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Product FAQ

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

Answer

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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Product FAQ

I performed stable selection but my antibiotic-resistant clones do not express my gene of interest. What could have gone wrong?

Answer

Here are possible causes and solutions:

Detection method may not be appropriate or sensitive enough:
- 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.
- Insufficient number of clones screened: Screen at least 20 clones.
- Inappropriate antibiotic concentration used for stable selection: Make sure the antibiotic kill curve was performed correctly. Since the potency of a given antibiotic depends upon cell type, serum, medium, and culture technique, the dose must be determined each time a stable selection is performed. Even the stable cell lines we offer may be more or less sensitive to the dose we recommend if the medium or serum is significantly different.
- Expression of gene product (even low level) may not be compatible with growth of the cell line: Use an inducible expression system.
- Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest: Linearize the vector at a site that is not critical for expression, such as within the bacterial resistance marker.

Answer Id: E9183

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