To achieve accurate size and concentration results on the Fluidity One, your sample must not contain any primary amines, other than those in the protein of interest. This means that Tris buffer and free amino acids must be removed.
The following protocol for removing these species has been developed from extensive testing in our development lab to ensure reliable results every time.
Why do primary amine additives need to be removed?
The Fluidity One detects proteins after the microfluidic diffusional sizing step by the addition of an amine reactive dye. This latent dye is based on ortho-phthalaldehyde (OPA) and is fluorogenic, meaning that it only fluoresces following reaction with a primary amine group.
In most instances the dye reacts with lysine residues in the protein or peptide being tested, or with an unprotected N-terminus. However, if a sample contains other primary amines, such as Tris buffer or free amino acids, these are also labelled. This means the calculated size (hydrodynamic radius, Rh) is skewed by these much smaller species, and the results do not accurately reflect the protein of interest.
How was this protocol developed?
The protocol is the result of tests conducted in the Fluidic Analytics development laboratory, as described here. The data discussed here was obtained in developing the large sample volume (200 µL) protocol. Similar tests were performed to develop the small sample volume (30 µL) protocol, but the specific results are not discussed in depth here.
Commercially available Rabbit IgG and glycine were used to prepare a test solution (100 µg/mL IgG in PBS pH 7.4 with 250 mM glycine) and a control solution (100 µg/mL IgG in PBS pH 7.4 only).
The hydrodynamic radius (Rh) and concentration of each solution was tested using a Fluidity One. Tests were performed before any treatment, and following 1x, 2x, 3x and 4x sequential passes through 2 ml Zeba spin columns1, as described in the protocol.
The results are shown in Figure 1.
Figure 1: Results of A. Size and B. Concentration from Fluidity One analysis of the IgG solutions following treatment with spin columns. Results reported are an average of at least 2 repeats.
In the case of the no treatment sample, the presence of the added glycine at high concentration meant that the sample could not be read by the instrument. A “concentration too high” error was given, and no result could be reported.
The solution containing glycine is seen to have a much smaller measured Rh after one spin than the control solution. This suggests that there is glycine remaining in the sample, lowering the average Rh. After a second spin the observed size of the protein is consistent between the glycine containing and control solutions. The size then remains steady over the third and fourth spins. From this we observe that two treatments with a spin column are required to fully remove the glycine.
After a second spin the observed size of the protein is consistent between the glycine containing and control solutions. The size then remains steady over the third and fourth spins. From this we observe that two treatments with a spin column are required to fully remove the glycine.
In both solutions we can see the concentration decrease with each spin column treatment, as not all of the sample is recovered. The reduction after two treatments is around 50 %. For this reason, it is recommended that the initial sample is at least 20 µg/mL to account for losses during the buffer exchange protocol.
Is there a way to improve sample recovery?
The protocol for larger sample volumes (200 µL) involves passing the sample through a 2 ml Zeba spin column twice in sequence, which resulted in a 50 % sample recovery for our study.
Adding 40 µL of buffer when pipetting the sample onto the prepared column can improve sample recovery rates, but this is optional.
Do you have a protocol for smaller sample volumes?
Yes, a modified version of the protocol is available which only requires 30 µL of sample. See the protocol sheet for full details of this.
Can I discuss my results with you?
If you have any questions about this protocol, or your Fluidity One results and how to interpret them, contact us by email, phone or web chat and we’ll be happy to help.
1: Columns used; Zeba Spin Desalting Columns, 7K MWCO, 2.0ml
ThermoFisher Scientific, catalog No. 89890 (pk 25) or 89889 (pk 5)