western blot troubleshooting high background

When analyzing the results, variations between lanes regarding loading and transfer rates between blots, must be taken into consideration. The setup of the experiment can be varied in many ways to best suit the specific inquiry. The separation on the gel is not only due to size but also to some extent depending on the molecular charge, hydrophobic regions, and degree of denaturation. An image is taken of the membrane and the result is analyzed.īy adding a separate marker solution to one of the wells in the gel, it is possible to estimate the size of the protein in addition to the antibody interactions that are used to verify the specific protein.

To visualize the protein of interest the membrane is commonly first probed using a primary protein-specific antibody followed by a labeled secondary antibody used for detection. After the transfer, the membrane is blocked in order to prevent unwanted membrane-protein interaction in the following steps. In order to further analyze the proteins, they are transferred onto a membrane in a procedure called blotting. Samples are prepared and loaded on to a gel and during the electrophoresis the negatively charged proteins move toward the positively charged anode. The setup consists of a standard set of seven steps, Figure 1.įigure 1. Towbin et al described electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets where the original gel pattern was accurately obtained. Towbin et al in 1979 ( Towbin, Staehelin, & Gordon, 1979) and two years later given its name by W.

It is built on a technique that involves transferring, also known as blotting, proteins separated by electrophoresis from the gel to a membrane where they can be visualized specifically. Western Blot (WB) is a common method to detect and analyze proteins.

Western blot troubleshooting high background