The Protein Man's Blog | A Discussion of Protein Research

Isolating genomic and plasmid DNA for further investigation and downstream application (e.g. PCR, sequencing, etc.) requires totally different protocols. While isolating genomic DNA merely requires you to crack open the cell walls and purify the resulting sample, extracting plasmid DNA may be a bit trickier and more complicated than this. Here’s a rundown on how these techniques differ.

INTRODUCTION

When we speak of mitochondria, anyone with knowledge of life science could tell you of its presence in animal cells and absence in plant cells. However, it should not be forgotten that another kingdom shares this cellular powerhouse - kingdom fungi.

Commercially available affinity purification supports are designed on the principle of specific surface interactions among biomolecules, including, but not limiting to, antigen-antibody, enzyme-ligand and lectin-carbohydate. Affinity chromatography is one of the most efficient tools used for purification of biomolecules of interest, including proteins, glycoproteins, lipids and nucleic acids. In affinity chromatography, one of the interacting molecules is covalently bound to the resin and is addressed as a ligand. The stationary ligand bound resin interacts with the ligand interacting proteins or biomolecules, which are passed through the resin in mobile phase and hold them to the resin. These molecules are later eluted as a purified fraction with an elution buffer.

Good Western blot blocking methods are crucial in order to achieve clean and reliable results. Protein samples are first ran on a polyacrylamide gel to separate proteins by size. The proteins are then transferred to a nitrocellulose or PVDF membrane. A blocking agent is then used to prevent primary and secondary antibodies from binding to the membrane non-specifically (areas without the protein of interest). Proper blocking methods cover the membrane surface without interfering with bound protein; thus preventing non-specific antibody binding and non-specific signal (noise or background) due to the membrane’s high affinity for proteins.