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Why is my fluorescent protein so dim?

Cells typically need only low to moderate expression of a drug resistance gene to acquire resistance to the drug. For fluorescent proteins (FPs), in contrast, high levels of expression are required to obtain bright fluorescence signal, whereas low to moderate expression may produce only weak signal or signal under detectable threshold of the hardware. In addition, certain FPs (e.g. EBFP) are much dimmer than common FPs (e.g. EGFP), and are especially difficult to detect. In fact, some researchers had to resort to immunofluorescence with anti-FP antibodies in order to visualize their FPs of interest in cell culture or in vivo. More details on the use of FPs and their comparison to other reporters can be found in our VectorAcademy post here. Below are some of the most frequent causes for poor fluorescence:

The FP gene is driven by a weak promoter

Some promoters are inherently weak (e.g. UBC), and can result in low expression of the FP. Other promoters (e.g CMV) may work well in cell culture but can sometimes get silenced in vivo. When expressing FPs, you should always try to use a strong ubiquitous promoter (e.g. EF1A or CAG) if you can. If you have to use a tissue-specific promoter, you should also choose a strong one when possible. If you have to use a weak promoter or a promoter that is poorly characterized, you should be prepared for the possibility of inadequate FP signal. When this happens, it does not mean that your FP is not expressed. It may just be that it is not highly expressed. You can always check FP expression using more sensitive assays such as RT-PCR or immunofluorescence.

Read more about our collection of promoters

The FP gene is expressed from lentivirus or MMLV

For retroviral vector systems (e.g. lentivirus or MMLV), internal polyadenylation signals cannot be present between the LTRs, as this would inhibit virus packaging. Instead, a single polyadenylation signal is present in the 3’LTR. As a result, transcription from the upstream promoter often continues past the end of the upstream ORF, through the downstream promoters and ORFs. This often leads to partial inhibition of expression of the downstream ORF(s). When the downstream ORF is an FP, then its expression can be much reduced. You can improve your FP gene expression as follows:

The FP gene is expressed in a polycistron

When the FP gene is expressed in a polycistron, there are a number of complications as described below, and sometimes the expression of the FP gene is dependent on the upstream or downstream ORF partners.

  • The FP gene is downstream of an IRES.

    In a polycistronic transcript containing one or more IRES elements, the ORF downstream of the IRES is expressed at much lower levels (typically 10-20%) as compared to the upstream ORF in the polycistron. If an FP is expressed downstream of an IRES, this reduction in expression may well lead to poor fluorescence. If you have to express an FP in a downstream position of a polycistron, you can consider using a 2A self-cleaving linker (e.g. P2A or T2A) instead of IRES. By using 2A, the downstream ORF of a polycistronic transcript is usually expressed at a level that is comparable to (or only moderately lower than) the upstream ORF. However, 2A also has its pitfalls that could comprise function of the gene of interest (GOI). When multiple 2As are present, the downstream ORFs also tend to be expressed at lower levels. Additionally, 2A self-cleavage is not 100% efficient, and the efficiency can be strongly influenced by the sequence context of the upstream and downstream ORF. As such, a significant fraction of the translation product from the polycistron could be fusion protein that has failed to self-cleave, which could be a concern in some applications. Furthermore, the cleavage of the 2A linker leaves behind an extra short peptide on the C terminus of the upstream protein and an extra proline on the N terminus of the downstream protein. This is not an issue in most applications, but under some circumstance, it can comprise protein function.

    Learn more about IRES vs. 2A linker in polycistronic expression cassette

  • The FP gene is part of a fusion protein.

    Fusion proteins almost always show weaker fluorescence than their unfused counterparts (e.g. EGFP/Neo fusion protein has weaker fluorescence than EGFP alone). Also, untested fusion proteins have the potential to be unstable, misfolded, or nonfunctional within the cell. If you suspect that this is the reason for weak fluorescence, you can try the following:

    • Consider using a stronger promoter (e.g. EF1A) to drive expression of the fusion gene.

                      Read more about our collection of promoters

    • Consider expressing the fluorescent protein in an unfused form.

    • Consider using a brighter FP variant (e.g. using TurboGFP instead of EGFP).

                      Read more about our collection of fluorescent proteins

    • Consider adding a linker, or use a different linker than what you already use, to connect the FP with your GOI.

                      Read more about our collection of linkers

    • Consider switching the position of the FP and GOI.

The inherent brightness of the FP gene is low

Some FPs are inherently dimmer than the other variants in the same color family. For example, in blue color family, the brightness of EBFP is only about one third that of TagBFP. We suggest that you choose a brighter FP variant based on our guide on fluorescent reporters.

Read more about our collection of FPs and how they compare to each other

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