Dot Blot Analysis

Background information

Dot blot

The Dot blot technique is a very simple method used to analyzing, detecting and identifying proteins. It is very similar in principle to the detection of samples in Southern blot, Northern blot, or Western blot. In the dot blot assay, the protein samples are not first separated by electrophoresis. Instead, the protein samples to be detected are spotted through circular templates directly onto the paper substrate or membrane.

The Dot blot methodology offers significant savings in time, the technique can be done less than an hour as the procedures for the gel such as gel electrophoresis and chromatography are not required. However, due to unavailable information on the size of target molecules. The dot blot

The Cu1+ then react to bicinchoninic acid assay forming a purple water soluble complex. Moreover, the total volume of protein concentration can be measured by the colorimetric technique which will change the colour of sample solution from green to purple in proportion to protein concentration.

SDS-PAGE electrophoresis

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE electrophoresis) is probably the most common analytical technique widely used to separate biological molecules, usually a nucleic acid or protein based on their electrophoretic mobility. The motility is a function of conformation, the length of their peptide chain and charge of the molecule. Depending on their size, small biomolecules move faster and more easily fit through the pores in the gel than larger ones. Therefore, biomolecules are separated according to their size.

Dialysis

The technique is often used in research to detect specific proteins which have extracted from cells. In this process, a mixture of proteins separated based on two distinguishing properties which are molecular weight and antibody binding specificity. According to the procedure, proteins first separated based on size which have to perform with SDS-PAGE. Next, the proteins from the gel are then transferred to a polymer membrane (PVDF or nitrocellulose) to make them more accessible to the antibodies that specific to the target protein.

Cytotoxic assay

Cytotoxicity is described as the quality of being toxic to cells. The technique is used to measuring dead cell protease activity, in which they have lost membrane integrity. There are several ways used to measuring cytotoxicity, but generally involved in the assessment of cell membrane integrity. Vital dyes such as propidium iodide or trypan blue can be used to estimate the membrane integrity by measuring the ATP content or protease biomarker with the MTT redox potential assay. Many of these assays are involved in colorimetric or fluorescence detection.

Purification by cobalt affitynity