- Proteins and Peptides
- Lysates and Cell Lines
Antibodies bind to specific sequences of amino acids, known as the epitope. Because amino acid sequences are different from protein to protein, antibodies can recognize specific proteins among a group of many. Therefore, a single protein can be identified in a cell lysate that contains thousands of different proteins and its abundance quantified through western blot analysis. First, proteins are separated from each other based on their size. Second, antibodies are used to detect the protein of interest. Finally, a substrate that reacts with an enzyme is used to view the antibody/protein complex.
Next, the protein concentration is determined. Loading buffer, which contains SDS (sodium dodecyl sulfate), and BME (beta-mercaptoethanol) is added to the protein suspension.
The sample is then heated to near-boiling, which denatures the protein and allows the SDS to bind to the protein. SDS carries a negative charge.
Once the sample(s) and ladder are loaded, a current is run across the gel, with a negative pole on the well end of the gel, and a positive pole on the opposite end of the gel. Because the protein is bound to negatively charged SDS, it is pulled down through the gel to the positive pole. The larger the protein, the slower it moves.
Although running the gel is the last step in the SDS-PAGE method, it is important to make note of several pieces of information:
After the gel is run, it is placed against a membrane, and current is passed across the gel to the membrane, transferring the proteins onto the membrane.
The transferring method detailed in this How To Guide is known as semi-dry, however transferring can also be done using the wet method. The membrane is usually made of PVDF or nitrocellulose, both of which have advantages and disadvantages that should be throughougly researched prior to use.
The first step in immunoblotting is to wash the membrane and block it with non-specific protein. The non-specific protein binds to the surface of the membrane where protein is not already present. This will prevent antibody from binding to the membrane and giving a non-specific signal.
Next, the primary antibody is added to the solution in which the membrane is floating. Remember that the primary antibody recognizes a specific amino-acid sequence of a particular protein.
After a wash is conducted to remove unbound primary antibody, secondary antibody is added. Secondary antibody recognizes the primary antibody, and usually is conjugated with an enzyme, such as HRP (Horse Radish Peroxidase).
Lastly, another wash is performed to remove unbound secondary antibody. Non-specific binding of both the primary and secondary antibodies can occur, but thorough washing usually minimizes this problem. The amount of time the primary and secondary antibodies are applied, directly affects the specificity and strength of binding.
Once the substrate has been added, the light being emitted can be detected with film or a photo imager.
Notice that the film does not contain information on where the lit bands are located in relation to the membrane. Therefore, it is very important to use a method that allows the film to be aligned with the membrane in the exact same manner as when the film was exposed. The membrane is usually stained after the detection step, so that the protein present can be visualized, and compared to the film.