Having difficulties with your experiment?

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View the relevant questions below:

 

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Reporter Vectors
I used one of your pTracer™ vectors and do not see any fluorescence. What could have happened?

Here are possible causes and solutions:

  • High background fluorescence due to riboflavin in the culture medium—replace medium with 1X PBS to eliminate background fluorescence.
  • A filter set was used that did not allow excitation at the optimal wavelength or permit detection of the emitted fluorescence—use the XF76 filter from Omega Optical.
  • Transfection efficiency is too low to allow detection of transfected cells—optimize your transfection conditions or try another method.
  • Expression of Cycle 3 GFP may be low depending on the cell line used—in HEK-293 cells, maximum fluorescence was observed 72 hours post-transfection.
Specialty Vectors
I used your pDisplay™ vector to express my protein on the cell surface. The protein is expressed based on a western using anti-HA antibody, but is not displayed on the cell surface based on immunofluorescence using anti-myc antibody. Can you please help troubleshoot?

We would recommend checking to make sure that the gene of interest is cloned in-frame with the Platelet-derived Growth Factor Receptor (PDGFR) transmembrane domain.

I used the pSecTag2 vector and am seeing cellular but not secreted expression of the protein of interest. Can you offer some tips?

If the gene of interest has a start codon in the context of a perfect Kozak sequence, it maybe preferentially translated over the vector’s initiation codon, resulting in no leader sequence and no glycosylation, and hence no targeting to the endoplasmic reticulum and no secretion. This is rare, but it has been observed. If it is a problem, we recommend using PCR to delete the start codon.

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