How To Achieve High-Quality Western Blots

1 How To Guide How To Achieve High-Quality Western Blots Guy Regnard, PhD Western blotting, or immunoblotting, is an essential tool in any molecular laboratory. This sensitive assay can identify specific antibodies or antigens, having applications in both research and clinical laboratories.1 Western blotting typically uses antibodies to recognize short linear regions of amino acids found in the denatured protein of interest.2 For example, the assay can identify infection by detecting specific antigens in a patient’s serum; alternatively, it can also detect antibodies produced in response to a disease or vaccination. Generally, the first step in western blotting is to denature and separate proteins using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). After that, these proteins are electrophoretically transferred onto a membrane. Primary antibodies are used to probe the membrane and bind to target proteins. Secondary antibodies conjugated to enzymes then bind to the primary antibodies. The final step involves visualizing the protein band using chromogenic or chemiluminescent substrates that are cleaved by enzymes, or fluorescence using fluorophores that are attached to the secondary antibody. Optimizing each step is crucial—this guide summarizes how you can achieve high-quality western blots. Protein isolation and sample preparation Throughout the western blotting process, it is crucial to ensure that you use deionized, distilled water. This is particularly important as any changes in pH can affect the outcome at each stage.3 When isolating your proteins, first check whether the extraction buffer is compatible with western blotting. Extraction buffers that contain high salt (>250 mM) or nonionic detergent (>2%) concentrations or acidic/basic pH buffers can affect the buffering system of the gel, causing poor protein resolution.4 Endogenous proteinases are the second aspect of protein isolation that can affect western blotting. Protein extraction from samples often releases endogenous proteinases, which, if left unchecked, can rapidly degrade your sample. Fortunately, a few extra steps will mitigate their impact. These include adding proteinase inhibitor to the extraction buffer, maintaining samples on ice to reduce proteinase activity and immediately inactivating proteinases after adding sample buffer by heating samples to 95°C for at least five minutes.2,3 It is helpful to quantify the protein concentration in each sample using a standard protein assay. Quantifying protein concentration allows you to dilute or concentrate your samples to prevent overloading or underloading of the SDS-PAGE gel.2 HOW TO ACHIEVE HIGH-QUALITY WESTERN BLOTS 2 How To Guide Proteins are separated according to size when using SDS-PAGE. This separation relies upon the complete denaturation of the protein and ensures protein migration and resolution.4 Negatively charged SDS molecules bind the length of the protein, giving the protein a negative charge proportional to its size. Therefore it is important to add sufficient sample buffer to ensure an excess of SDS, which prevents the inherent charge of the protein from affecting mobility.2 Your protein samples are now ready for gel electrophoresis. Separating your proteins When designing a western blot experiment, ensure that any samples you want to make statistical comparisons are loaded on the same blot. Given the variability between western blots, statistical comparisons between blots are not advisable.5 Before loading your SDS-PAGE gel, a 2-minute centrifugation at 17 000 × g removes any insoluble material in the sample that can cause streaking within the gel or affect how the samples run.2 Dedicate a lane on the SDS-PAGE gel to a pre-stained protein standard; this will allow you to monitor the progress of the gel electrophoresis visually.1 Furthermore, the pre-stained protein standard can indicate whether transfer of your proteins onto the membranes has occurred during the blotting process. Molecular weight markers are also an essential control element. In addition to the molecular weight marker, it is important at this stage to consider incorporating positive and negative controls to electropherize alongside your sample. These controls will be important during analysis and are discussed in greater detail later in the guide. Transferring your proteins Always use gloves when preparing transfers as oils on the hand can interfere with the transfer process.3 Separated proteins can be transferred from the gel onto a membrane using wet or semi-dry blotting systems. The membranes most commonly used for this purpose are nitrocellulose and polyvinylidene fluoride (PVDF).4 Before the transfer, allow the gel to equilibrate in the transfer buffer for 30 minutes. This is particularly important when wet blotting as it prevents the gel from changing in size during the transfer process, which can cause a blurred transfer pattern. When assembling the transfer, make sure that there are no air bubbles, as these will block current flow and obstruct protein transfer in that area. For wet blotting, the best way to minimize air bubbles is to assemble under transfer buffer. With semi-dry blotting, air bubbles can be gently rolled out of the assembly using a clean glass rod. With semi-dry systems, cut the filter paper to the size of the gel. This ensures the current flows through the gel and does not bypass it through overlapping filter paper.3 Most proteins are negatively charged in the transfer buffer and will move towards the positive anode during transfer; however, some can become positively charged. In these instances, placing an additional membrane on the cathode side of the gel will capture any positively charged proteins, particularly those of interest. Wet blotting can be helpful, especially when transferring hydrophobic proteins or proteins greater than 100 kDa, as it allows for prolonged transfer without the risk of buffer depletion. As the transfer process generates heat, transfers longer than an hour should be cooled to between 10–20°C. Generally, the buffer temperature should not exceed 45°C, irrespective of transfer time. Once the protein transfer is complete, reversible Ponceau S staining can visualize the success of the transfer onto the membrane before the blocking step.3 HOW TO ACHIEVE HIGH-QUALITY WESTERN BLOTS 3 How To Guide Blocking is essential to prevent the primary antibody from binding non-specifically to the membrane resulting in a background signal during the detection step. Nitrocellulose membrane has a particularly high protein-binding affinity. The membrane can be blocked using a buffer containing reagents such as bovine serum albumin (BSA), gelatin, casein, nonfat dry milk and Tween 20. Blocking reduces the background signal during detection, thereby enhancing assay sensitivity.1 Some primary antibodies, particularly those raised in goats, can cross-react with proteins found in milk. If so, switch to an alternative blocking reagent to reduce the background.4 Optimizing your antibodies It is important to note that antibodies that recognize epitopes based on tertiary conformation may not bind or react weakly to denatured proteins bound to the membrane.1 If identifying tertiary structure is required, non-denaturing conditions can be used during gel electrophoresis; however, this will impact the mobility of the proteins. The optimal primary and secondary antibody concentrations should produce a low background and high specificity. These concentrations can be determined empirically using a dilution series with membrane strips.3,4 Additional protein bands, aside from the protein of interest, can often be detected when using polyclonal antibodies as the primary antibody. These are the result of contaminating antibodies present in the polyclonal antibody solution. Performing an analogous adsorption step before using the primary antibody can help remove contaminating antibodies.4 For example, an immune serum that detects a recombinant vaccine antigen made using a microbial expression system may detect multiple protein bands on a western blot. Preincubating the immune serum with a membrane coated in protein isolated from the microbial system can remove contaminating antibodies from the primary antibody incubation step. Nonspecific binding of the primary or secondary antibody can cause high background. To determine whether the problem exists with the primary antibody, controls using pre-immune serum or only the secondary antibody can be tested. If the primary antibody is the cause of the high background, changing the protein-blocking agent or lowering the primary antibody concentration can reduce the background and increase specificity.3 Once the primary and secondary antibodies are bound, the protein bands can be visualized. Detecting your proteins Protein can be visualized using chromogenic and chemiluminescent substrates or fluorescence using a fluorophore. The most cost-effective methods involve chromogenic substrates, which produce an insoluble precipitate on the target protein band upon cleavage by horseradish peroxidase or alkaline phosphatase-conjugated to the secondary antibody. Luminescent substrates can be used to improve the sensitivity of these conjugated secondary antibodies. Furthermore, using avidin-biotin coupling to a biotinylated secondary antibody can achieve extremely high sensitivity as multiple enzymes are attached to each antibody.3 An essential aspect of chromogenic substrates is that the reaction must be stopped to avoid background development and signal saturation.4,5 It is helpful to keep this development period constant over experiments and projects. A constant development time can allow for the early detection of problems with substrate and secondary antibodies routinely used in the laboratory. HOW TO ACHIEVE HIGH-QUALITY WESTERN BLOTS 4 How To Guide As with optimizing antibodies, the linear detection range is empirically determined. This is especially important when performing quantitative analysis of western blots to ensure the signal generated by the conjugated enzyme is proportional to the protein concentration.6 Regarding analysis, it is essential to discuss all immune-positive protein bands. State reasons behind excluding any protein band from the analysis.5 Controls, controls, controls Controls are crucial when performing western blotting, not only to confirm the success of the blot but also to support the conclusions reached based on the results obtained.5 For example, suppose you want to detect a protein in a heterogeneous mixture of proteins. In that case, dedicating lanes of the SDS-PAGE gel for both positive and negative protein controls is helpful. A positive protein control will enable you to confirm that the western blotting has succeeded and that the primary and secondary antibodies are functional. A negative protein control will indicate any nonspecific binding of the primary antibody to proteins other than the target or whether the protein target is present irrespective of the experimental conditions. An additional control to consider is excluding the primary antibody from the assay. Excluding the primary antibody will indicate whether the secondary antibody non-specifically binds to the membrane or protein bands. Controls should also include pre-immune sera.5 When quantifying bands, including a loading control will ensure that observed differences in protein band intensity are not due to differences in loading volume or total protein between samples. A helpful checklist detailing controls is available here. 5 Conclusion Given the scope for detecting proteins and their sizes, the above steps will undoubtedly assist you and your lab in producing publication-ready western blots. Sponsored by: HOW TO ACHIEVE HIGH-QUALITY WESTERN BLOTS 5 How To Guide References 1. Sam-Yellowe TY. Exercise 13: Immunoblotting (western blotting). In: Immunology: Overview and Laboratory Manual. Springer. Published online 2021:335-341. doi: 10.1007/978-3-030-64686-8_37 2. Kurien BT. Western Blotting for the Non-Expert. Springer; 2021. doi: 10.1007/978-3-030-70684-5 3. Gallagher S, Winston SE, Fuller SA, Hurrell JG. Immunoblotting and immunodetection. Curr Protoc Cell Biol. 2011;52(1):6-2. doi: 10.1002/0471143030.cb0602s52 4. Litovchick L. Immunoblotting. In: Greenfield EA ed. Cold Spring Harbor Protocols. Cold Spring Harbor Laboratory Press. 2020;2020(6):pdb-top098392. doi: 10.1101/pdb.top098392 5. Alexander SP, Roberts RE, Broughton BR, et al. Goals and practicalities of immunoblotting and immunohistochemistry: A guide for submission to the British Journal of Pharmacology. Br J Pharmacol. 2018;175(3):407. doi: 10.1111/bph.14112 6. Pillai-Kastoori L, Schutz-Geschwender AR, Harford JA. A systematic approach to quantitative western blot analysis. Anal Biochem. 2020;593:113608. doi:10.1016/j.ab.2020.113608