Applications in Blotting
Blotting reagents are essential in western, northern and southern blotting for sensitive and consistent detection of proteins and nucleic acids from complex samples. These reagents improve the efficiency of electrophoresis and, with the help of antibodies, enable radiometric, chemiluminescent, chromogenic, and fluorescent detection for use in the fields of molecular biology and immunogenetics.
Western blotting separates proteins on a gel by electrophoresis before transferring them to a membrane, upon which the proteins are detected by chemiluminescence, fluorescence or colorimetry.
See how Western Blotting is being used in breakthrough research in our article: Are Antibody Vaccines the answer to Alzheimer's Disease?
Southern Blotting uses electrophoresis to separate DNA fragments which are transferred to a positive nylon membrane.
Northern Blotting separates RNA fragments via electrophoresis before transferring the resultant bands to a positively charged nylon membrane.
Blotting Reagents Products
Blotting Reagents for these processes include:
Transfer buffers help shift the sample over from the gel used in electrophoresis to the detection membrane.
Wash and Blocking Buffers
Wash buffers are repeatedly used during the blotting process. For example, in western blotting, between antibody incubation steps, wash buffers are used to remove any excess antibodies between primary and secondary antibody incubation steps. During northern and southern blotting, washing buffers remove unhybridized probes.
Blocking buffers, on the other hand, prevent the antibodies from nonspecific binding.
Filter / Blotting Paper
The filter paper is necessary for use in the transfer sandwich to enable quick and efficient transfer by supporting wicking and uniform capillary action, as well as preventing contamination.
The blotting membrane (used for detection) consists of either nitrocellulose or PVDF (polyvinylidene fluoride). Both membranes are suitable for use in Western Blots, while PVDF is also capable of binding nucleic acids well, so it can be used in northern, southern and dot blots as well. However, northern and southern blotting also use positively charged nylon membranes, which can permanently bind nucleic acids with low background and high target retention.
Detection reagents include chemiluminescent substrates, chromogenic substrates and fluorescence detection kits. All these reagents interact with a probe on the antibody bound to the protein of interest (western) or a hybridization probe (northern/southern) to provide a visual signal.
Chemiluminescent substrates have the greatest sensitivity, able to quantify concentration and can be used after reprobing. They require the use of an x-ray film and dark room to detect the light emission created by a reaction between two solutions.
Chromogenic substrates do not need any visualization equipment because they form an insoluble colored precipitate on contact with the appropriate enzyme. However, this method lacks sensitivity.
Fluorescent kits can quantitatively analyze a blot without a darkroom or film and can be used to target multiple, different samples on the same blot without reprobing.