Details

Autor(en) / Beteiligte

• Ortega Ibarra, Jorge M.
• Cifuentes-Castro, Víctor H.
• Medina- Ceja, Laura
• Morales-Villagrán, Alberto

Titel

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

Ist Teil von

• Journal of neuroscience methods, 2021-07, Vol.358, p.109194-109194, Article 109194

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2021

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• [Display omitted] •This procedure permits the quantification of several proteins in a single sample.•This technique uses small volume of sample saving reagents used in immune detection.•Several proteins can be monitored in a single experimental session.•The results showed here are in accordance with previous data obtained by other methods.•These results confirm the role of interleukins during seizures and epilepsy. 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. 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. 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. 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.