Fluorescent ligand binding - frequently asked questions

Useful answers to frequently asked questions and troubleshooting advice for customers using fluorescent ligand products.

  1. What does the product look like?
  2. How should I reconstitute? The fluorophore is red, but the solution is blue - have I made it up properly?
  3. What excitation and emission wavelengths and filter sets should be used?
  4. Cells don’t bind the fluorescent ligand
  5. Cells label everywhere (cytoplasm, nucleus), not just the membrane
  6. Control (non target-expressing) cells label as well as the target cells
  7. There is very high background fluorescence
  8. Cells label well, and show a good saturation-binding curve – but the maximal saturation binding level is lower than expected
  9. There are bright fluorescent particles in the cell cultures

1. What do the fluorescent ligand products look like?

Appearance:
The products are supplied as a dried film of blue material adhering to the inside of the conical bottom of a ‘champagne flute’ brown glass vial. The product should be visible as a dark smear through the side of the vial.

2. How should I reconstitute the fluorescent ligand? The fluorophore is red, but the solution is blue - have I made it up properly?

Check the individual datasheet for reconstitution details. Generally, we recommend the following:

If dissolved in DMSO at 10mM according to the instructions in the datasheet, the product will appear to be a dark blue solution.

For sub-aliquoting in slightly larger volumes, the product can also be reconstituted at 1 mM by adding 10x the volume of DMSO stated.

The product can be further diluted in DMSO, for example to  100 μM, and will retain its blue colour.

Final dilution to ~ 100 nM is in buffer (+ 0.1% DMSO)

3.  What excitation and emission wavelengths and filter sets should be used?

For emission, use a narrow-band filter with a peak at 650 nm.
Shorter wavelength excitation and longer wavelength emission filters can be used but signals will be weaker, and there may be cross-talk with other fluorophores.

 

Each fluorescent binding ligand product is provided with a datasheet that recommends the appropriate excitation wavelength and emission filter-set for optimal fluorescence detection of the product.

4. Cells don’t bind the fluorescent ligand

Likely causes:

  1. Cells are not expressing the receptor
    • Compare with cells from another source
    • Compare with cells at different growth stages
    • Use the ligand for FACS to select cells expressing the receptor
  2. The receptor is not reaching the cell membrane
    • Ligands do not readily cross the cell membrane, so this is difficult to test
    • An antibody approach with fixed, permeabilised cells might confirm this
  3. The receptor is unable to bind the ligand.
    • Fixing the cells can prevent ligand binding – use live cells
    • Too much solvent (DMSO) in buffers can damage the cells

5. Cells label everywhere (cytoplasm, nucleus), not just the membrane

This is likely to be because the cells are not healthy and have taken up the ligand non-specifically

  • Check the culture conditions – cells should not be over-confluent
  • Check the assay conditions – pH should be 7.4 in isotonic buffer (e.g. HBS or PBS)
  • Ensure that minimal solvent is used (e.g. DMSO < 0.1%)
  • Check the cells have not been fixed and permeabilized
    Even in a healthy culture, there will be dead cells that show non-specific staining
  • Make sure cell-plates are handled carefully and removed from the incubator immediately before use, not left sitting on the bench for long periods

6. Control (non target-expressing) cells label as well as the target cells

Likely cause: high non-specific binding to control and target cells

  • Ensure that both control and target cell lines are healthy (see previous FAQ)
  • Ensure the ligand is not being used at too high concentration. (The maximu should be 100nM)
  • Check if the ligand is binding to an endogenous receptor expressed by the host cells by blocking with an appropriate unlabelled competitor

    • Cross-reaction to other receptors

  • Abcam provides some cross-selectivity data, but the ligands may bind to other receptors
  • Use the pharmacology of the unlabelled drug from which the ligand is derived as a guide to potential additional targets to which the ligand may be binding
  • Test this with selective unlabelled blockers for the off-target receptor

7. There is very high background fluorescence

BODIPY-630/650 should not show this because it’s fluorescence is quenched in aqueous solution. If this is reported, it is likely to be due to a high concentration of the ligand in solution around the cells.

  • Use the ligand at a lower final concentration (30 – 100nM gives the best signal : background)
  • Add a brief (10 min), gentle wash step to reduce the background fluorescence

8. Cells label well, and show a good saturation-binding curve but the maximal saturation binding level is lower than expected

This may be due to the fluorescence detector reaching saturation, rather than the cells reaching binding saturation.

  • Try reducing the gain on the detector
  • Try taking measurements on an alternative instrument

9. There are bright fluorescent particles in the cell cultures

Explanation: microscopic crystals of ligand have precipitated out of solution onto the cells.
Likely cause: the ligand is not completely dissolved in DMSO before use.

  • After thawing the DMSO stock aliquot of ligand, sonicate it briefly to ensure the ligand is dissolved and the solution is homogeneous
  • Then, dilute the ligand to 2x final concentration in buffer for addition to an equal volume of assay buffer covering the cells
  • Do not repeatedly freeze/thaw the stock ligand aliquots – it can cause crystals to form
  • Do not freeze/thaw the final ligand-buffer solution – this can also cause crystals to form

Abcam products:

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