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Choosing Buffers for FCS Assays

Although it is of primary importance to select buffers compatible with the biological and chemical interactions to be studied, you must also meet requirements for effective FCS measurements.

Important Buffer Properties to Consider
  • pH: In addition to affecting the interactions and stability or your biological probes, buffer pH can affect the function of many fluorescent dyes. (See Choosing Fluorescent Dyes for FCS Assays).
  • Salt Concentration: Salt concentrations can affect the stability and aggregation state of your probes. Additionally, high salt concentrations can be detrimental to some fluorescent dyes, such as Quantum Dots.
  • Detergents: Detergents can limit the amount of aggregation of your probes and reduce adsorption to the chamber, but can also affect fluorescent signal intensity and diffusion rates.
  • BSA: Adding BSA to your buffer can stabilize your biological probes, but may interfere with interactions or affect diffusion rates.
  • Other Additives: Any other additive must be measured to ensure it does not contribute significant background fluorescence to your sample.
List of Buffers Used in Published FCS Assays
  • PBS + 0.05% v/v Tween-20 (Casein cleavage and IL-4 receptor)
  • 100 mM diethanolamine-HCl (pH 10) and 1 mM MgCl2 (alkaline phosphatase hydrolysis)
  • 50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 10 mM MgCl2, 10 mM DTT and NH2-sulfate (p21 ras nucleotide exchange)
  • 20 mM HEPES (pH 7.4), 50 mM NH4Cl, 0.05% Tween20, 10 mM MgCl2 (RNA-ligand binding assay)
  • 50 mM Tris-HCl (pH 7.2), 10 mM MgCl2, 0.05% Tween20 (tyrosine kinase assay)
  • 50 mM MOPS (pH 7.5), 1 mM EDTA and 0.1% TX100 (trypsin and chymotrypsin assays)
  • 600 nM substrate, 1-2 uM enzyme in 50 mM HEPES (pH 7.5), 30% sucrose, 150 mM NaCl, 0.1% Tween20, 0.01% PEG3350 (CMV protease assay)
  • 50 mM MOPS (pH 7.5), 1 mM EDTA, 0.1% TX-100 with reaction termination by dilution 100-fold with 300 nM avidin in same buffer (leader peptidase assay)
  • 140 mM NaCl, 20 mM HEPES (pH 7.4), 5 mM MgCl2, 1.8 mM CaCl2, 0.36 g/L NaHCO3, 1 g/L D-Glucose (EGF binding to EGF-R in vesicles)
  • 60 mM KCl, 5 mM MgCl2, 10 mM HEPES (pH 7.9), 250 mM ZnSO4, 2 ug/reaction poly-DC with 6.8 nM oligonucleotide and 0.3 to 3 ul nuclear extract (protein-DNA interaction…transcription factor)
  • 60 mM KCl, 5 mM MgCl2, 10 mM HEPES pH 7.5 (thyroid hormone receptor/DNA interaction)
  • 0.18M NaCl (DNA hybridization studies)

See FCS References for a list of published FCS experiments.