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

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.

Both protoplasts and spheroplasts refer to altered forms of plant, bacterial or fungal cells from which the cell wall has been partially or completely removed. These cells usually have all the other cellular components, except for the cell wall. When used in reference to bacterial cells, protoplasts may also refer to the spherical shape assumed by gram-positive bacteria while spheroplasts refer to the spherical shape assumed by gram-negative bacteria upon partial or complete removal of the cell wall. Cells with compromised cell walls assume a characteristic spherical shape to better withstand the rigors of its surrounding environment. They are also extremely sensitive to osmotic and mechanical shock.