western blotting

Western Blotting

A core technique in cell and molecular biology

Loading Control Antibodies - Generate publication quality Western blots

Loading Control Antibodies - Generate publication quality Western blots

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Western blotting, also known as immunoblotting or protein blotting, is a core technique in cell and molecular biology. In most basic terms, it is used to detect the presence of a specific protein in a complex mixture extracted from cells. Our Introduction to Western Blotting guide is the ultimate resource regarding this technique, answering all the questions you may have. It is also available in a downloadable pdf format.

Three Key Elements

The Western blotting procedure relies upon three key elements to accomplish this task:

  • the separation of protein mixtures by size using gel electrophoresis;
  • the efficient transfer of separated proteins to a solid support;
  • and the specific detection of a target protein by appropriately matched antibodies.

Once detected, the target protein will be visualized as a band on a blotting membrane, X-ray film, or an imaging system. Since Western blotting is accomplished rapidly, using simple equipment and inexpensive reagents, it is one of the most common laboratory techniques. The results achieved are also easy to interpret, unique, and unambiguous. Therefore, it is routinely used on its own, or along with other immunoassays, in research and clinical settings.

Why use a Western Blot?

Western blots are in wide use across a broad range of scientific and clinical disciplines. Their ability to clearly show the presence of a specific protein both by size and through the binding of an antibody makes them well-suited for evaluating levels of protein expression in cells, and for monitoring fractions during protein purification . Likewise, they are helpful for comparing expression of a target protein from various tissues, or seeing how a particular protein responds to disease or drug treatment.

In many cases, Western blots are used in combination with other key antibody based detection techniques, such as ELISAs or immunohistochemistry.

Western Blotting Antibodies

Antibodies selected for immunodetection should be Western blot tested if possible, with attention paid to the experimental conditions recommended by the antibody supplier. Usually, Western blot positive antibodies recognize a short linear sequence of amino acids found within the target protein that remains intact, or becomes visible, when the target protein is fully unraveled. This is because most Western blots are carried out under denaturing and reducing conditions which remove all higher order protein structure .

In contrast, some epitopes can be conformational, forming a three dimensional structural configuration of amino acids that will be lost upon denaturation of the protein . Thus, not all antibodies work in a typical Western blot . Since Western blot procedures allow for flexibility in choosing gel electrophoresis and blotting conditions, it is possible to modify buffers to retain enough higher order protein structure for detection by some antibodies . 

Bio-Rad is one of the world’s leading manufacturers of primary and secondary antibodies for Western blot anaylsis. In addition, we offer ISO-certified pre-made buffers to simplify immunostaining and make results more consistent from blot to blot. Our 100% Satisfaction Guarantee is backed by our quality, which makes ordering from Bio-Rad totally risk free!

Western Blot Tips

Our tips include a guide for the detection of low abundant proteins by western blot.

Western Blot Protocol

Our western blot protocol covers a general Western blot procedure for use with the majority of Bio-Rad reagents.