Nano dot blot: An alternative technique for protein identification and quantification in a high throughput format

Abstract

Background: Dot blot technique has been used in a similar way to western blotting, with the major difference being the lack of protein separation with electrophoresis. Protein samples are spotted over a membrane paper, the identification and quantification of a protein is achieved by immunodetection procedures such as colorimetry, fluorescence or chemiluminescence. This technique is widely accepted, but it uses large amounts of sample and antibodies to reveal the presence of the target protein. Significant milestones have been reached to achieve better results with the use of less sample and reagents; however, the ninety-six-well format is still in use.

New method: In this work, we propose an innovation to this technique, reducing the amount of sample and antibodies to identify a specific protein when compared to the regular dot blot method. Procedure consists of using a sample volume of approximately 200 nanoliters deposited with a multineedle device developed by our group.

Results: Five samples of standard protein or antigen can be spotted in a Cartesian format to identify and quantify the protein involved in physiological or pathological conditions. In addition, at least five replicates of sample or antigen are used to enable better statistics to calculate the concentration of every standard and the protein present in a sample.

Conclusions: Hundreds of samples can be deposited in a few minutes and analyzed in a single experimental session. To validate this method, which we called nano dot blot, six proteins involved in the inflammation process were tested in acute and chronic rat models of seizures.

Keywords: Cytokines; Epileptic biomarkers; Immunodetection; Inflammation; Nano dot blot; Seizures.