The control experiment performed on a biotinylated antibody, as a proof of concept, demonstrates the high efficiency of this method for depleting the target analyte. The amount of adsorption is calculated by comparing the target peak area of each sample with the untreated antibody (Figure S1
). One minute incubation results in 95% reduction of the target band. At 5 minutes and above, 99% depletion was observed. After establishing the high efficiency of our method, the performance of this technique was tested in a more complex system of bacteria lysate (as used in blotting experiment). Cells expressing Vasa-FLAG protein have several major protein bands, including one at approximately 36 kDa, which is the predicted mobility of the DNA-encoded construct. By performing the traditional (Western) immunoblotting, it was verified that the 36kDa band is the FLAG-tag protein (: lane 1–3 under immune-blot). An alternative approach for this assay is to use a protein chip following elution of proteins from beads that had been pre-incubated with the lysate. This approach was recently demonstrated [12
] but still required several hours to be accomplished with multiple manual steps. In the case of the queried bacterial bands here within whole cell lysates, a pre-absorption approach might be sufficient to rapidly detect the band of interest using minimum reagents as demonstrated in our method.
Samples were analyzed on an electrophoresis gel. (lanes 2–5) clearly shows the depletion of two bands at 12.4 kDa and 36.3 kDa for four incubation times. In comparing the intensities of the two bands in lanes 2–5 with the lysate, the target band is detected by the significant depletion of the 36.3 kDa band. The presence of the 36.3 kDa band in the elution sample (lane 6) not only confirms the presence of the target, but also the effective adsorption of the FLAG proteins on the beads. Even at one minute incubation, a significant depletion of the target protein is clearly observed. This suggests that the time scales for diffusion td and reaction tr for transport and adsorption of FLAG protein were in the range of minutes.
For detection and quantification of the depleted FLAG protein the electropherograms of lysate, 1 and 60 minutes samples are compared in . Even though one minute incubation is sufficient for the detection of target band on the gel (), the depletion continues at longer incubation times. However, the rate of depletion decreases after 10 minutes of incubation time (embedded graph in ). This is evident as rate of diffusion transport decreases with decrease in concentration gradient between the bulk and the surface.
Figure 2 Detection of FLAG protein (A), comparison of two incubation times in depleting the target (1.42 μg/μl after depletion with anti-FLAG magnetic beads. The arrow indicates the target peak. The adsorption kinetic of FLAG protein is shown in (more ...)
In order to compare our method with the elution technique, the electropherograms of supernatant and eluted protein after 60 minutes incubation are compared in . The presence of a distinct peak at 36.3 kDa, overlaying the 60 minutes target peak, confirms the eluted protein as the FLAG protein. Quantification of depleted and eluted FLAG protein was performed using the peak area (including the non-specific adsorption) and is listed in .
Quantification of the immunodepleted FLAG protein. The listed values are the average of three independent experiments (mean ± standard deviation).
(column 5) shows more than 66% adsorption of FLAG protein in the first minute of incubation and that the adsorption of FLAG protein gradually increases, from 66% to 82% after 59 minutes of incubation. The non-specific adsorption of 12.4 kDa band is also shown (column 3) with approximately constant adsorption (43%) in all of the samples. Since the 12.4 kDa peak appears far away from the 36.3 kDa FLAG protein peak, the non-specific binding does not affect the quantification of the target protein. However, the sensitivity of the assay would be limited in cases of interference of the target peak with other protein peaks in the sample. It is important to note that peak interference is a challenge in all the current methods available. More investigations are needed to improve resolution of the peaks by choosing different gel properties [16
], or changing the script of the system. Release of only 19% of the FLAG protein from the beads shows that even though the target can be detected after elution, and further improvements are possible by optimizing the elution process, an accurate quantification is not possible by this method.
Finally, the dynamic detection range of the target (~50kDa FLAG) was determined in a protein complex at different concentrations. The complex sample consists of four other proteins, each at 200ng/μl. The target electropherogram peak is shown in for one of the sample runs. All the peaks can be easily identified arriving at 35.5 seconds. As shown in (inset), the fluorescent intensity is linearly proportional to the target concentration.
The effect of non-specific binding of other proteins on the detection of the target was also investigated by electropherogram peak analysis. These electropherograms are shown in a supplementary figure S2
. shows the percentage adsorption of each protein in the complex sample. It is important to note that, even though the most significant depletion (95%–100%) was observed in the target band, other proteins in the complex were also depleted (less than 40%) through non-specific adsorption. Our results suggest that even at very low concentration of target (50ng/μl) and in case of non-specific binding of other sample components, the rapid and high specific detection of the target can be achieved.