Western blotting, also known as immunoblot is a technique that is used to analyse, detect and measure either a particular protein that is obtained w
Western blotting, also known as immunoblot is a technique that is used to analyse, detect and measure either a particular protein that is obtained within a complex mixture of either cells or tissue lysate (Singh et al. 2021). It allows researchers to study protein expression, interactions, and modifications in a sample. By separating proteins based on size, transferring them to a membrane, and using antibodies to identify the target protein, researchers can gain valuable insights into various biological processes. This report will discuss the necessary components and the technique involved for Western blotting.
Cell lysates are commonly used for this technique, allowing measurement of protein content in the cytosol (Taylor et al. 2022). Sample concentrations are determined using a spectrophotometer or assays like Lowry/Bradford (Begum et al. 2022). After reaching the required volume, samples are diluted with a loading buffer (e.g. Laemmli) to aid loading into gel wells. Tracer dye (e.g. bromophenol blue) is added for separation visualisation (Janes 2015).
After preparing the samples its vital that there are two types of control of samples, a positive control and negative control. A positive control can involve a known target protein source, like purified protein or control lysate, this confirms the protein identity and antibody activity and a negative control such as an untreated cell extract can act as a zero baseline to confirm nonspecific staining (Hidayat and Wulandari 2021). An additional purpose of these controls is to differentiate and determine specific characteristics of the results by removing the background noise which allows validation of the results.
SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) is a step in the Western blotting technique where proteins are denatured and separated. An anionic detergent bind to the proteins before electrophoresis (Nowakowski et al. 2014). The gel used in SDS-PAGE has two functions: stacking and resolving. The stacking gel concentrates and adjusts pH for clear bands, while the resolving gel separates proteins by pore size (Begum et al. 2022). This gel-based approach in Western blotting enables detailed analysis of protein size, polarity, and detection.
In this stage, proteins undergo electrophoretic transfer from gel to adsorbent membrane, known as electrophoresis (Hidayat and Wulandari 2021). Its vital that the membrane must be positioned in a way that lets negatively charged proteins move from the gel onto the membrane. This transfer can be completed in either wet or semi-dry conditions. Though, wet conditions are known to be more dependable specifically for larger proteins, as they prevent the gel from drying out (Hidayat and Wulandari 2021).
Following protein transfer, Coomassie R-250 or Ponceau S dyes are commonly used for staining (Goldman et al. 2016). GAPDH and actin act as control proteins in Western blotting, enabling comparison, validation, and standardisation of protein levels. Total protein staining (TPS) is a cost-effective alternative for visualising transferred proteins (Begum et al. 2022). Blocking buffer plays an important role in sensitivity, reducing background interference, and improving signal-to-noise ratio. The choice of blocking agent varies depending on the antibody-antigen pair (Hidayat and Wulandari 2021). Following blocking, the membrane is incubated with a primary antibody, washed, and then a secondary antibody with a signal-generating enzyme is added. Imaging captures the generated signal upon enzyme-substrate reaction (Begum et al. 2022).
There are many factors that can affect the experiment results, these issues can be include: weak or unusual bands may result from non-specific antibody binding, no bands could be due to technical errors like nonsterile equipment or insufficient transfer time will result in weak signal or no intensity (Bass et al. 2016). Faint bands or weak signals can arise from low protein concentration, high background bands or smudges could be caused by non-specific antibody binding or inadequate washing steps, and even or uneven spots on the stains may be due to uneven protein loading or transfer issues (Yang and Mahmood 2012). To ensure reliable results, it is important to be able to identify and resolve all the issues that may arise during the experiment.
Western blotting technique is used to detect specific proteins, the results are often displayed in images with a grey background. When analysing these images and comparing the protein bands to a standard set, an important consideration is the identification of the target protein (Kroon et al. 2022). If a band is present, it indicates the protein's presence, while the absence of a band suggests either its absence or low levels. The intensity of the bands can also be measured to determine the protein's concentration (Pillai-Kastoori et al. 2020). Several statistical tests, such as t-tests, ANOVA, and non-parametric tests, are applied to determine if there are any significant differences between the samples. Control samples are a key factor in ensuring accuracy by accounting for variations in protein levels (Pillai-Kastoori et al. 2020). By comparing the results, researchers can identify patterns or differences in protein expression levels. The interpretation of the results depends on the specific purpose of the experiment (Pillai-Kastoori et al. 2020).
In summary, Western blotting is a simple cost-effective technique that is able to be a versatile in many key areas such as detecting proteins, measuring levels, studies modifications and interactions, identifies disease-related changes, and ensures quality control in biologics. It provides valuable insights in research, aiding diagnostics, treatment development, and protein analysis across diverse experimental conditions.
References:
Bass, J.J.,, Wilkinson, D.J.,, Rankin, D.,, Phillips, B.E.,, Szewczyk, N.J.,, Smith, K., and Atherton, P.J., 2016. An overview of technical considerations for western blotting applications to physiological research. Scandinavian Journal of Medicine & Science in Sports, 27(1), pp.425.
Begum, H., Murugesan, P., and Tangutur, A.D., 2022. Western blotting: A powerful staple in scientific and biomedical research. BioTechniques, 73(1), pp.5869.
Goldman, A.,, Harper, S., and Speicher, D.W., 2016. Detection of proteins on blot membranes. Current Protocols in Protein Science, 86(1).
Hidayat, R., and Wulandari, P., 2021. Western blotting (WB) technique guideline for separation and isolation of protein. Bioscientia Medicina: Journal of Biomedicine and Translational Research, 5(4), pp.367380.
Janes, K.A., 2015. An analysis of critical factors for quantitative immunoblotting. Science Signalling, 8(371).
Kroon, C.,, Breuer, L.,, Jones, L.,, An, J.,, Akan, A.,, Mohamed Ali, E.A.,, Busch, F.,, Fislage, M.,, Ghosh, B.,, Hellrigel-Holderbaum, M.,, Kazezian, V.,, Koppold, A.,, Moreira Restrepo, C.A.,, Riedel, N.,, Scherschinski, L.,, Urrutia Gonzalez, F.R., and Weissgerber, T.L., 2022. Blind spots on western blots: Assessment of common problems in western blot figures and methods reporting with recommendations to improve them. PLOS Biology, 20(9).
Nowakowski, A.B., Wobig, W.J., and Petering, D.H., 2014. Native SDS-PAGE: High resolution electrophoretic separation of proteins with retention of native properties including bound metal ions. Metallomics, 6(5), pp.10681078.
Pillai-Kastoori, L., Schutz-Geschwender, A.R., and Harford, J.A., 2020. A systematic approach to Quantitative Western blot analysis. Analytical Biochemistry, 593, p.113608.
Singh, K.K., Gupta, A., Bharti, C., and Sharma, H., 2021. Emerging techniques of western blotting for purification and analysis of protein. Future Journal of Pharmaceutical Sciences, 7(1).
Taylor, S.C.,, Rosselli-Murai, L.K.,, Crobeddu, B., and Plante, I., 2022. A critical path to producing high quality, reproducible data from quantitative western blot experiments. Scientific Reports, 12(1).
Yang, P.-C., and Mahmood, T., 2012. Western blot: Technique, theory, and trouble shooting. North American Journal of Medical Sciences, 4(9), p.429.