blotting technique 1- Vertical electrophoresis 2- Membrane blotting 3- Membrane blocking 4- Washing steps 5- Incubation with 1ry antibody overnight 6- Washing steps 7- Incubation with 2ry antibody for about 1h 8- Washing steps 9- Membrane development and signal detection Membrane blocking Western blotting involves the immobilization of biomolecules on a membrane via hydrophobic interactions. As non-specific binding of antibodies to the membrane is detrimental to the specificity and sensitivity of the assay, it is essential to "block" spaces not already occupied by proteins. Your choice of blocking strategy will be guided by your samples and the antibodies used. As a starting point for choosing blocking agents, buffers and optimal conditions, begin by using those recommended by the manufacturers of the detection reagents. Two main classes of blocking agents are commonly used for Western blotting: A- proteins B- non-ionic detergents A- Proteins as blocking agents They are known as permanent blocking agents as they form a physical attachment to the membrane. Requirements of blocking agents: 1- It is critical that the blocking agent has a greater affinity for the membrane than the antibodies used in subsequent steps. 2- the blocking agent should fill all unoccupied binding sites without disrupting binding interactions between transferred proteins and the membrane. The most common permanent blocking agents include bovine serum albumin (BSA), non-fat milk, normal goat Serum and casein B- Detergents as blocking agents
Detergents inhibit non-specific hydrophobic binding of
proteins to membranes. They are considered non permanent blocking agents since they do not attach to the Membrane and can be removed in a simple washing step. A solution of Tween-20 is commonly used, but alternatives may be considered, such as Triton X-100, sodium dodecyl sulfate (SDS) or NP40, which are especially useful to counter strong background signals. Detergent blocking agents are usually used in conjunction with protein blocking agents. Antibody probing
Once your protein samples are separated and transferred
onto a membrane, the protein of interest is detected and localized using a specific antibody. Usually, Western blotting protocols utilize a non-labeled primary antibody directed against the target protein and a species-specific, labeled secondary antibody directed against the constant region of the primary antibody. Primary antibody probing
Following the blocking step, the protein of interest can
be detected using antibodies. Special considerations concerning 1ry antibodies: 1- If you purchase a commercially available primary antibody, ensure that it has been validated for Western blotting applications according to the manufacturer. 2- You can save money by reusing the primary antibody solution, but be careful how you store it so that it retains its activity. 3- Use clean tubes and store refrigerated when not in use 4- You can also consume less primary antibody by incubating the membrane in a smaller box. 5- If the protein of interest is well characterized i.e. if you know its molecular weight and where it is expected to migrate on the membrane, cut the membrane to the appropriate smaller size after transfer. Washing step After primary antibody probing, it is necessary to wash the membrane in order to remove excess antibody that could cause high background and, consequently, a low signal-to-noise ratio. A low concentration detergent solution, such as 0.05 to 0.1% Tween-20 in PBS or TBS is commonly used, especially after incubation with highly concentrated antibody solutions or crude extracts. PBS-Tween is suitable for most washing applications. However, TBS-Tween is preferable where the target proteins are phosphorylated, as the phosphate in PBS may interfere with antibody binding. 2- Secondary antibodies
A wide variety of secondary antibodies are commercially
available and your choice will depend firstly on the species in which the primary antibody was produced. If, for example, the primary antibody was of the IgG isotype and produced in goat, the secondary antibody must be an anti-goat IgG antibody produced in another species. The procedures for incubation of the secondary antibody solution and the membrane are essentially similar to those described for the primary antibody. Role of secondary antibody: 1- secondary antibody serves as a carrier of the label. 2- is also a mechanism to amplify the emitted signals, as many secondary antibodies can theoretically bind simultaneously to the primary antibody. For this reason, secondary antibodies are most often polyclonal and can target epitopes on the framework regions of the primary antibody; specificity is thus limited to species and immunoglobulin isotype. The signal emitted by the labeled secondary antibody is then measured and is proportional to the quantity of protein of interest present on the membrane. Detection step Chemiluminescence
The HRP-conjugated secondary antibody binds to the
primary antibody, specifically bound to the target protein on the membrane. After the addition of a luminol peroxide detection reagent, the HRP enzyme catalyzes the oxidation of luminol in a multi-step reaction. The reaction is accompanied by the emission of low intensity light at 428 nm. In the presence of certain chemicals, the emitted light is enhanced up to 1000-fold, making it easier to detect, and thus increasing the sensitivity of the reaction in a process known as enhanced chemiluminescence (ECL). Several enhancers can be used, but the most effective are modified phenols, especially p-iodophenol, which increases HRP turnover rate and assists in the transfer of electrons from luminol to the enzyme. The intensity of signal is a result of the number of reacting enzyme molecules and is thus proportional to the amount of antibody, which is related in turn to the amount of protein on the blot The emitted signals can be detected using X ray films Examples of signal detection using X ray films and ECL kit