#Steps of western blotting technique full

Rolling brush: exclude bubbles within the multilayer membrane.PVDF/NC membrane: receive the proteins.Filter paper: protect the gel and membrane, keep a wet and conductive condition.Sponges: protect the gel and membrane, keep a wet and conductive condition.Cassette: clamp the multilayer films when doing protein transfer.Power source: provide a constant electric field.Electrophoresis tank: used for holding the electrode chamber and anode buffer.Electrode chamber: used for holding the gel cassette and cathode buffer.Gel knife: help to move the gel from cassette.Gel comb: used for toothing one side of the gel as sample loading area.

Gel: usually made of polyacrylamide, need precast before western blot.

Here we list several frequently used tools and materials for western blot:

#Steps of western blotting technique full

The experimental equipment of western blotĪs western blot is a very mature technology in molecular biology, there are a full set of commercialized reagent and equipment that support the experiment of WB (Figure 3). To probe and visualize the separated protein bands. This procedure is basically an indirect ELISA process (know more about indirect ELISA at ELISA Guide).Ĭreative Diagnostics provides a variety of high quality primary antibodies and secondary antibodies for western blot use.įigure 2. After the proteins band transferred onto PVDF/NC membrane, we use anti specific protein primary antibodies to probe the sample protein, and then use a labeled secondary antibody for further visualization (Figure 2). When the proteins were successfully separated, we still use an electric field to transfer the protein bands onto another polyvinylidene fluoride (PVDF) or nitrocellulose (NC) membrane for the following detection. Protein electrophoresis: proteins separated under electric fields based on different molecular weight. These control proteins are commonly known as molecular weight marker or protein ladders.įigure 1. If we put several proteins with known molecular weight under the same electrophoresis condition along with the tested samples as control, we may further measure the molecular weight of proteins within the tested samples by compare the location of sample proteins with control proteins. If we load the mixed protein samples on one side of the gel, add a constant electric field on the gel, and let the protein migrating on the gel for a period of time, the mixed protein sample could be separated into different bands on gel (Figure 1). That's why we could separate those runner proteins. The bulky runner would be affected more by the 'branches' than the small runner so that they runs slower. This is just like the runners running through a jungle, the polyacrylamide molecule in the gel is like the branches along the sideline, which could slow the runner down. The migrating speed depends on the molecular weight of proteins: heavy proteins move slower than light proteins. Proteins are large molecules with charge, they can migrate in the polyacrylamide gels under electric field. Electrophoresis is a commonly used method for separating proteins on the basis of size, shape or charge. The basic principle of western blot are protein electrophoresis and ELISA. Through spatial resolution, this method provides molecular weight information on individual proteins and distinguishes isoforms, alternate processing products, and other post-translationally modified forms. Immunodetection involves the identification of a protein through its reaction with a specific antibody. And then transferred the protein sample to a membrane (typically nitrocellulose or PVDF), where they are probed with labeled-antibodies and stained with dyes for visualization and directly identified by N-terminal sequencing, mass spectrometry or immunodetection. It uses gel electrophoresis (usually SDS-PAGE) to separate native proteins by 3-D structure or denatured proteins by the length of the polypeptide. The bases of western blot identification are two distinguishing properties: molecular weight and antibody binding specificity. Eastern blot is used to analyze protein post translational modifications (PTM) such as lipids, phosphomoieties and glycoconjugates.ĭifferent from those blot technique described above, western blot, also called protein immunoblot, is an analytical technique used to detect specific proteins in a given sample such as cell lysates, tissue homogenate or extract. Southern and northern blot are nucleic acid blotting techniques, of which southern blot is for detecting a specific DNA sequence in DNA samples and northern blot is to study gene expression by detection of RNA (or isolated mRNA) in a sample. The principle and process of these blot techniques are similar. There are many 'Blot' techniques in molecular biology such as southern blot, northern blot, eastern blot and western blot. Antibody Incubation & Gel Visualization What is western blot