Western Blots Protocols

A Western blots are a type of protein blot that is used in the medical field, biology labs and other scientific disciplines on a daily basis. Western blots, when performed properly, are a way of studying protein expression in tissue samples. Because there can be differences in the parameters of each sample, there are a number of protocols that should be followed in order to obtain accurate results.

Performing Washes

• After incubating the membranes in the antibody solutions for the appropriate amount of time, which varies depending on which antibody solution is being used, EnCor Biotechnology states that the membrane should undergo three room temperature washes in TBS. TBS is a solution that has a specific makeup and pH for washing. The membrane should be washed in between each introduction of a new antibody solution.

Preparing Staining Mixture

• According to WesternBlotting.org, preparing the staining mixture immediately prior to applying it to the member will achieve the best results. There are multiple types of staining solutions, some of which can be purchased and some of which must be mixed in the lab. Chemiluminescence staining, according to EnCor Biotechnology, provides the most information per stain but is also the most expensive and requires a darkroom for developing. Horse Radish Peroxidase Staining and Alkaline Phosphatase Blotting are other options for staining. Regardless of which method is used, preparing the mixture just prior to use is essential.

Using a Buffer

• After applying the selected staining solution, the use of a buffer is key. In chemiluminescence staining, applying an acetate over top of the staining solution is protocol, according to WesternBlotting.org. Horse Radish Peroxidase Staining requires use of an ammonium acetate buffer in order to obtain the brown staining product. The buffer used in Alkaline Phosphatase Blotting, which should create a purple staining product, is more complicated to make and can be purchased already made.