BD Living Colors FAQ's - Troubleshooting

GFP
DsRed

Q I have trouble detecting GFP variant expression in transiently transfected cells. What can I do?

A

1. Verify that the detection method is appropriate in terms of filter sets.
2. If you have transfected a fusion construct, test the vector alone, to confirm that detection conditions are good.
3. If a fusion construct has been made, sequence the junctions in question to confirm that the fusion is in frame.
4. Test a different cell line with the construct. We typically use HeLa, COS, HEK293, CHO-K1, or NIH3T3 cells.
5. Try varying the conditions of transfection, e.g. amount of plasmid, tranfection reagent, and confluency of cells upon transfection.

Q How can I troubleshoot fluorescent protein expression in yeast?

A For EGFPs:

• Enhanced fluorescent proteins include human-codon optimization. In general, we recommend using the non human-codon optimized variants in yeast. However, we have anecdotal information from researchers which suggests that the mammalian variants will work in yeast. We offer non-human codon optimized Cyan and Yellow proteins.
• Preliminary studies suggest that the promoter used in yeast must be very strong for detection of GFP and DsRed variants. It may be that the proteins are being expressed at levels that can not be detected under the analysis conditions. If possible, use a strong constitutive promoter to optimize expression.
• If you are generating fusion proteins, try sequencing through the junctions in the construct to verify that the fusion construct is correct and in frame.

For multi-color labeling:

• Sequential transformations are recommended so that detection of the first variant can be confirmed before transformation with the second and/or third.

For detection with fluorescence microscopy or flow cytometry:

• Optimize detection with single filter sets for each individual variant before moving on to additional variants.
• Grow colonies on plates first, confirm fluorescence, then transfer to liquid. In bacteria, GFP variant fluorescence can be detected starting from overnight to 24 hours; DsRed fluorescence can be detected starting from from 36–48 hours.
• Perform Western analysis to determine if the proteins are present in the lysate. This helps to determine if the problem is in protein synthesis or activation for fluorescence. This is especially important if fusion proteins are being made.

Q How can I troubleshoot no or low signal on Western blots?

A

1. Try sonication for 30 seconds when preparing the lysate.
2. Try higher concentrations of the primary antibody.
3. Try loading higher quantities of lysate. We typically load 10 µl per lane of a 1 mg/ml total protein lysate.
4. Try a different antibody. Although the conditions for the Western are denaturing, it may be that the conformation of the fusion masks the epitopes.
5. It may be that fluorescence is detectable by confocal microscopy, but the fusion protein is not in high enough concentration to be detected by Western blot.

Q I selected GFP expressing stable clones that express stable clones and, after culturing, the fluorescence levels have decreased or become variable. What could be the reason for this?

A We don't have a direct explanation for this type of phenomenon. It is probably the case for any stably transfected gene. However, it is more explicit with the GFP due to the sensitivity fluorescence detection. There are a couple of possibilities:

1. The original single clone included more than one clone.
2. Even within a population derived from one clone, there may be variations within the population. During maintenance of the original clone with cells growing to confluency, and under selection pressure, cell to cell variation may develop.

Q I selected stable clones that express GFP and, after culturing for some time, the fluorescence levels have decreased or become variable. What can I do about this?

A

1. For transient expression, the plasmid, once it enters a cell, is episomally maintained. The cellular transcription and translation machinery is used to express the transfected gene of interest. Variations from one cell to the next can occur because multiple plasmids have been taken up by one cell. These cells would have higher expression levels since the copy number of the plasmid is higher. In general, for stable transfectants, only 1 or 2 copies are integrated. Therefore, stable transfectants will usually have lower expression levels than transient transfectants.
2. Determine whether the cells were near confluency or confluent at 48 hours after transfection or when selection pressure was initiated. Contact inhibition of growth can decrease transfected gene expresssion.
3. Verify whether the cells were healthy at 48 hours after transfection as well as during neomycin selection. If the cells were not healthy during the initial transient expression stage, this could be an indication of potential toxicity.
4. Perform a kill curve with the G418. If the concentration used for selection is too potent (potency can vary from lot to lot), the selection pressure could be too high. If the cells are under stress to survive due to high concentrations of selection agent, expression levels of reporter genes not directly linked to the selection antibiotic gene may be down-regulated. Effects on the morphology or healthiness of the cells may also become an issue. These effects become more pronounced with an extended exposure to high concentrations of antibiotic. For maintenance of post selection cultures, we suggest lowering the antibiotic concentration to between 1/2 and 1/5 of the selection concentration.
5. Generally, depending on how long the current cells have been in culture (if more than 1–2 months), we recommend thawing a fresh aliquot of the original stock to ensure a homogenous population.
6. It is possible to enhance your population of antibiotic resistant clones by sorting them through flow cytometry. This allows you to obtain a subpopulation of stable clones that express at a higher level. Sorting of clones can be done after the antibiotic selection procedure has been completed.

TROUBLESHOOTING for DsRed Variants

Q Why do I see aggregation or clumping in cells that express DsRed1?

A

1. The maturation time required for DsRed protein, as well as the turnover rate, appear to be longer for DsRed when compared to GFP variants. This may affect when fluorescence first appears in transfected cells as well as the localization and aggregation of the protein.
2. Since the DsRed protein cells takes longer to fold and be processed, some of the clumping and aggregation may be partly due to the fact that the protein is being detected during its processing through the ER, Golgi, transport, etc.
3. If a fusion construct has been used, the interaction of the two proteins involved in the fusion may also affect the final folding and function of the fusion protein. Because DsRed is not mutant variant of the GFP, the same procedures that have been used for GFP variants may not always give the same type of results with DsRed.

Q What can I do to decrease aggregation in cells that express DsRed1?

A

1. If possible, express the fusion in a different cell line. We typically use HeLa or HEK293.
2. Express the fusion from a weaker promoter or transfect less plasmid, especially if aggregation or mislocalization due to protein build-up is a problem in that particular cell line.
3. Transfect more plasmid if sensitivity is an issue.
4. Insert the gene of interest at the other terminus if a fusion is being constructed.
5. Analyze the cells at 48–72 hours instead of 24–48 hours.
6. If stable cells are being selected, start antibiotic selection at least 72 hours after transfection. The initial seeding density should be such that the cells do not grow to confluence before the start of selection.
7. Add a linker or spacer sequence to the region between the DsRed1 and gene of interest. We have anectdotal evidence which suggests that GFP fusions require additional distance between the two proteins to help folding and localization

Q I am having trouble selecting stable DsRed1-expressing cells using cloning discs and antibiotic selection. What can I do?

A The lower intensity of DsRed1 hinders selection of clones from cloning discs. We suggest using a cell sorter (FACS) to sort the most intensely fluorescent cells into a 96-well plate. Alternatively, consider DsRed2, which exhibits increased fluorescence intensity.