Western Blot (Protein Immunoblotting) is a routine method to detect and analyze proteins. However, precautions and tips should be taken if you want to get reproducible and reliable blots.
At Sino Biological, we have over ten years of experience in Western Blot and have developed a set of tips and tricks to ensure robust data generation and cleaner blots.
Although general guidelines for protein loading and antibody dilution are recommended by the literatures and antibody manufacturers, the relative abundance of the protein of interest and the titer of the antibody used sometimes require further optimization of those parameters. Generally, 1 μg of purified protein or 10 μg lysate containing the protein of interest should be enough to be detected by a solution containing 1 μg/mL of primary antibody. Nevertheless, for low abundance proteins in a cell lysate might require as much as 50 μg of total protein and at least 2 μg/mL of antibody. Conversely, high background or undesired cross-reactivity also can be modulated by adjusting these parameters in the other direction.
For most proteins can be successfully transferred by applying ~14V overnight in a wet transfer system or a maximum current of ≈0.8 mA/cm2 of gel area in a semi-dry system. While some proteins requiring improved transfer efficiency onto the membrane include large proteins exceeding 100 kDa or very hydrophobic proteins. They can be subjected to extended transfer times at high power using the semi-dry system but will require cooling to keep a constant transfer temperature of ~20°C. Also, the transfer buffer can be modified to increase transfer efficiency by adding SDS at a concentration of 0.1% (w/v). If using nylon membranes, SDS and methanol should not be used.
Gel thickness has a double effect in immunoblotting, influencing both quantity and quality of antigen detection. Generally, the thickness and acrylamide percentage of the gel inversely correlates with protein transfer efficiency and band diffusion, with gels 0.5– 0.75 mm transferring more efficiently than thicker 1.0–2.0 mm gels. Also, the protein bands from thinner gels usually resolve better and provide crisper, well-defined detection.
Always remember to equilibrate the membrane 10 to 15 min in transfer buffer before transferring. Because PVDF membranes are hydrophobic and will not wet from just being placed into transfer buffer, first immerse 2s in 100% methanol, then equilibrate 10 to 15 min with transfer buffer. If the membrane dries out, wet again with methanol and then transfer buffer. Following electrophoresis, we recommend to equilibrate the gel 30 min at room temperature in transfer buffer to prevent a change in the size of the gel during transfer. Changes in gel dimension usually result in a blurred transfer pattern.
One of the most critical parameters to obtain clean western blots is the choice of an appropriate blocking agent. Blocking solutions work better when supplemented with a mild detergent like Tween-20, usually between 0.05% and 0.5% (v/v). A number of blocking agents can be used and these include immune analytical grade non-fat dry milk powder, BSA fraction V or normal animal serum at working concentrations ranging between 0.5% and 5% (v/v). Serum may be the best solution for problematic backgrounds, apparently by reducing unspecific interactions between the primary antibody and the blocking agent. When using serum, it should be from the same species as the primary antibody or from the same species as the secondary when secondary antibody detection is used.
The potency of a primary antibody might be leveraged by optimizing the incubation time with the antigen. Usually, one hour incubation should be enough to visualize the protein of interest, however, overnight incubation at 4⁰C will be an enough time for the antigen-antibody reaction to occur and result in detection of a positive signal. One hour incubation at room temperature is usually enough for the conjugated secondary antibody and certainly, it should not be more than three hours since it might generate high background during detection.
Usually, gel electrophoresis is under non-denaturing conditions when you have your antibody working in other immunoassays but not in western blot. Since proteins are usually separated under denaturing conditions during gel electrophoresis, this limits the detection of proteins by antibodies recognizing structural epitopes in non-denatured proteins.
Some of the factors should be considered when performing immunoblotting with phospho-specific antibodies are buffer compatibility, antibody specificity and protein abundance.
The using of Blotto or other blocking mixtures containing dry milk is unsuitable because phospho antibodies could bind to a number of protein components in milk. Instead consider choosing BSA-based or alternative blocking buffers. A common problem related to phospho detection is that the phospho antibody is unable to detect the low abundant proteins in a cell lysate. This problem can be overcome by means of immunoprecipitation as described in tip9.
Detection of low abundant proteins by western blot can be achieved by immunoprecipitation of the target protein using a specific immunoprecipitation antibody, more protein of interest to be loaded in the sample lane. Depending on the amount of lysate used in the immunoprecipitation, stronger signal can be achieved by this method. Since the target protein can co-migrate with the heavy or light chain of the immunoprecipitation antibody that will react with the HRP-conjugated secondary antibody and obscure the signal from the protein of interest, it is necessary to use a qualifying reagent.
When the same membrane is required for testing of several proteins using different antibodies, stripping and re-probing is always possible although it might need to be empirically optimized for a particular assay since each antigen-antibody interaction is always distinct. In general, stripping buffers are reagents that combine low pH, detergents, reducing agents and/or heat in order to remove residual antibodies. Although repeated re-probing can lead to loss of signal, several re-probings are generally possible. Biotin-streptavidin interactions cannot be dissociated by this method but the whole complex can be removed away from the bound protein on the membrane.