How to perform BSA vs Fluorescein Titration

Introduction

Flow-Induced-Dispersion-Analysisis a capillary-based microfluidic method, where flow rates in the center of the capillary are faster than at the edges of the capillary. The resulting radial concentration gradients at the front and the tail of the sample zone result in the diffusion of your indicator. The distinct dispersion profiles determine the shape of the generated curves and enable a “first principle” biophysical measurement of size. The FIDA size measurement is broadly applicable for studying biomolecular stability, interactions, etc.

Serum albumins are very well studied proteins and widely available. Albumin is an important protein with many physiological functions such as maintaining osmotic pressure and carrier for many small molecule drugs and proteins. Albumins are also known to bind to dye molecules with low affinity. There have been several publications which characterise binding of fluorescein to BSA in low micromolar range. Binding of BSA and fluorescein can serve as a good practice in obtaining affinity constants for this interaction.

Material

BSA, Fluorescein, PBS, MillQ water, NaOH (1M).
96 well pressure plate with plate seal (220-010).
Standard Capillary (100-001).
Vial set, pkg of 200 pressure vials (including caps and inserts) (210-200).

Instrument

Fida Neo with 480-detector.

Instructions

Launch Fida Neo data acquisition software and place the two trays in sample load positions, either by opening and closing the door or by going into the manual control and choosing sample load under inlet positions and pressing apply (Figure 1).

Figure 1: Manual Control: Inlet Position set to Sample Load in Tray 2

Prepare the following solutions:

750 µM BSA in PBS. Dissolve 49.8 mg of BSA (MW=66.463 kDa) in 1µL of PBS.
1 M NaOH, I µL
1.25 µM Fluorescein in PBS, 100 µL

Prepare three vials with no inserts (Figure2), and pipette 1 µL of NaOH, PBS, and milli-Q water into separate vials. Label them, snap the pressure cap tightly and insert them in tray-2 in positions 1, 2 and 5 respectively, according to the tray 2 vial map (Figure 3).

Interaction between BSA and fluorescein is low affinity, in low µM range, therefore the premix assay format is recommended. Prepare a plate map according to Figure 4. In the first column, analyte column, going from bottom to top direction, deceasing concentrations of BSA. Highest concentration of BSA at the bottom in H1 should be 750 µM, with one to three dilutions going up, and A1 should be PBS only, i.e., zero concentration of BSA. Minimum of volume in each well is 51 µL, allowing for three replicates, each replicate requires 12 mL plus 15 µL for dead volume (3x12=36, 36+15=51). The second column, indicator column, should have the same concentrations of BSA as the first column, in addition, each well should have a 50 nM of fluorescein. Minimum volume is each well is equal to the dead volume, 15 µL.

Protocol for generating analyte (BSA) dilution and indicator (fluorescein) plate.

A. Add 200 µL of PBS buffer is each well in column 1, A1-G1, except H1. Add 300 µL of BSA, 750 µM, into H1 (Figure 5).

B. Take 100 µL from H1 and add to G1, mix by pipetting up and down three times. This is the first one to three dilution. Repeat dilution from G1 to F1, F1 to E1, and up the column till B1. Skip A1 as it is zero concentration of BSA.

C. Remove 100 µL from B1 to get 200 µL.

D. Using a multi-channel pipettor, take 48 µL from each well in column 1 and pipette into corresponding well in column 2, i.e., A1 to A2, B1 to B2 etc.

E. Since analyte concentrations in column 2 will have been diluted by 4%, after addition of 2 µL of fluorescein in the next step, using a multi-channel pipettor, take 6 µL from a PBS buffer and deliver to column 1 A-H to match the concentrations in the analyte wells and indicator wells.

F. Add 2 µL of 1.25 µM of fluorescein into each well in column 2 to give a final concentration of 50 nM of fluorescein in each well ([2x1250]/50=50 nM). Mix by pipetting up and down three times.

It is informative to run both CapMix and Premix for the same interaction to ensure that both assay formats give similar results. You will find both methods described below. In general, PreMix assay gives better results as compared to CapMix. However, often the two assay formats give similar results which indicate the k_on is fast and it is appropriate to continue to use CapMix.

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Writing a PreMix Method

A. Place the pressure seal on top of the 96-well plate and seal all the wells by pressing firmly on the sealer against the plate until all wells are pressure sealed. Place the plate in tray 1, ensuring that A1 is closest to the edge of the instrument, lower left corner of the tray 1.

B. Standard capillary method (Figure 6), by default, will take analyte and indicator from tray 1 and NaOH and assay buffer from tray 2. It does awash with 1 M NaOH followed by assay buffer prior to picking up analyte and indicator to ensure that the capillary is clean prior to running the assay. It picks up NaOH from tray 2, vial 1 position and assay buffer from tray 2, vial 2 position.

Figure 6: Standard capillary method, NaOH and buffer are in tray 2 while analyte and indicators are in tray 1

C. In Fida Neo software, click sequence, and then click add a line or double click in the first row of cells to generate the first assay. By default, the software will generate an assay with generic indicator name with indicator and analyte positions at 1 and 11 and waste position at 1 in triplicates (Figure 7), using standard capillary method. Standard capillary method is what we would be using in this assay.

D. Double click on the first cell under sample name and change it to 50 nM Fluorescein + BSA. Change indicator vial position in A2 and analyte vial position to A1. Double click on the analyte concentration and change it from nM to µM from the drop-down menu (Figure 8).

Figure 8: Modifying sequence for BSA vs fluorescein

E. Add seven more line by clicking on “Add line” (Figure 9).

F. Single click on first cell under indicator and drag down to change indicator vial positions automatically from A2 to H2. Repeat the same for analyte vial positions to change them from A1 to H1. Under analyte concentrations, double click on the last line and change it to 728, then change line seven to 242, then single click 728 and drag up to second line to fill in concentrations automatically (Figure 10).

Figure 10: Full sequence with eight concentrations

G. Save the data in a folder by clicking on the folder icon. You can also save the sequence in the same folder for future use (Figure 11).

H. Close the door and wait for the capillary light on Fida 1 to go green and then click “start sequence”.

Data Analysis

Launch Fida data analysis software, click on Data Analysis at the top. You can adjust the parameters such as placement of cursers, baseline drift, standard versus minimal fitting etc.

A. Load data by clicking on the icon for “Load data files”, navigate to folder where data was saved in and load all the data.

B. Click one data at a time, start by selecting the fluorescein alone or [BSA]=0 first, adjust the curser positions as necessary. If there is adrift in the baseline, click on “Baseline drift” for automatic correction. Select “single species” fit for analysis. The Hydrodynamic radius of fluorescein should be around 0.6 nm. Press “Save” to preserve the fit (Figure 12 and 13).

Figure 13: Determine hydrodynamic radius (Rh) for each concentration

C. Repeat steps above for all datapoints. Note that at 728 µM of BSA, viscosity is no longer equal to viscosity of pure water, but difference is small and can be ignored for this practice.

Figure 14: Model selection and binding curve.

D. On top right-hand corner, click ‘’Generate binding curve’. In the binding curve window, click on “Fit binding curve”. Default stoichiometry is 1:1 (Figure 14 and 15). Inspect the hydrodynamic radium for all the data point and if there are outliers, either remove by highlighting the outlier and then clicking “Delete selected” or go back to the data analysis and refit after making some adjustments such as curser placement. An outlier could be due to air in the capillary or insufficient filling of the capillary.

Figure 15: Plot of Rh vs BSA.
Apparent size of fluorescein increases as a function of BSA concentration.

E.Since fluorescein fluorophore is sensitive to its environment, its fluorescence is quenched upon binding to BSA. The quenching effect could be plotted as well by clicking on the box next to “Plot fluorescence” (Figure 16).

Figure 16: Plot of fluorescence vs BSA. Total fluorescence of fluorescein decreases as a function of BSA concentration. The environment of fluorescein changes from aqueous to hydrophobic, i.e., PBS to protein.

F. You can save data analysis by clicking on “Generate report”. By default, the report is saved in the same folder as that of raw data. Affinity of BSA to fluorescein is around 20-40 µM in this assay format.

A CapMix Method

Capillary mix (CapMix)is a shortcut method compared to the premix method. In this method, indicator and analyte are not premixed prior to injection into the capillary. Mixing occurs inside the capillary. Since there is approximately ten-fold dilution of the indicator plug, due to dispersion into analyte, there will be sufficient mixing of indicator and analyte prior to reaching the detector, which takes about 110 seconds. If k_on is fast i.e., less than one 60 seconds, this method gives similar results to the PreMix method. This method is faster, simpler, and consumes less indicator and analyte reagents. In this method, since there is no need for premixing, only one indicator well is needed (Figure17).

Protocol for CapMix plate

Follow the same protocol as premix plate except only one indicator well is needed. Make analyte dilution the same as premix plate.

Writing a CapMix Method

Capmix method will be very similar to the premix method with exception that all indicator vials will be the same, I.e., A2. Follow the same steps as the premix method except that in step 6, leave all indicator wells as A2. Analyte vials should be the same as premix, i.e.A1-H1.

Data Analysis

Data analysis for CapMix assay is the same as PreMix. Follow the same steps.

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