Benefits of iFluor labeling:
While there are many fluorophores available for antibody labeling, not all are created equal. GenScript offers labeling with iFluor dyes as they have superior properties compared to alternatives including:
- Greater photo-stability: The energy used to excite a fluorophore and drive subsequent light emission, can also destroy fluorescence. Known as "photobleaching," prolonged exposure to light chemically destroys the dye through non-reversible cleavage of covalent bonds. This can be problematic for repeated imaging of samples, or during extended periods of sampling such as those involved in time-lapse imaging. iFluor chemistry allows for durable fluorescence due to improved resistance to photobleaching compared to alternative dyes of comparable wavelengths.
- Stronger absorption and fluorescence: Fluorophore performance depends upon the ability to absorb and emit the greatest amount of energy within its intended wavelength. iFluor commonly outperforms other fluorophores emission strength, making for extremely sensitive and robust detection of your labeled antibody.
- Resilient signals in varying pH: Harsh environments such as lysosomes can degrade typical fluorophores, negatively impacting their performance. iFluor pH-insensitive dyes offer optimal signal detection from pH 3 to 11.
When using fluorophores, individually or in a multiplex format, it is important to pay attention to a few technical features including:
- Instrument capabilities:
Before choosing fluorophores, confirm the specifications of the equipment that will be used to measure or visualize the appropriate wavelengths. Excitation and emission energies of fluorophores should match the laser and/or filter combination energy as closely as possible. Depending on the generation of your equipment, wavelengths may be predetermined or selectable to within ±2.5 nm range.
- Wavelength overlap:
When using multiple fluorophores, maximal separation between each excitation and emission wavelength will result in the least bleed-through (cross-emission, cross-excitation, emission/excitation overlap) from one fluorophore to the next. Signal amplitude may also create this unwanted overlap and should be taken into consideration. If overlap is unavoidable, prioritize separation of excitation wavelengths.