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

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.

Lytic enzymes such as cellulase, lysozome, labiase, achropeptidase and a number of others, play a significant role in protein and DNA extraction. While different lytic enzymes can be used for different applications, cellulase is usually the enzyme of choice when it comes to breaking down plant cell walls. What is it about cellulase that makes it particularly useful for plant cell lysis? Here are some things you should know.

Commercial stripping buffers, such as G-Bioscience’s Western ReProbe™ and Western ReProbe™ PLUS, are specifically formulated to dissociate and remove all primary and secondary antibodies from the membrane-immobilized proteins without destroying the antigenic binding sites or removing the protein. This allows for several reprobings on the same membrane which will save you time, money, and precious protein samples. Western blotting can be time consuming (protein electrophoresis (1-2 hrs) + protein transfer (0.5 hrs to overnight)), so you want to make the most of your precious membranes and proteins by stripping and reprobing your nitrocellulose or PVDF membrane. There are many advantages to stripping your membrane such as: conserving limited/expensive protein samples, using different antibodies to analyze the same sample and confirm results, compare phosphorylated and total protein on the same blot, recoup antibody for later use, run less SDS-page gels and transfers, and to remove/adjust the concentration of your antibody.