Signal stability over time enables repeat exposures and the comparison of gels and membranes prepared at different times (Fig 3). The authors declare that there are no conflicts of interest that may have affected this study. Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Comparison of reflectance and transmission densitometry, using document and laser scanners, for quantitation of stained Western blots. Each individual sample as well as pooled samples was aliquoted and stored at -80C. values that were present in Figures 1(b) and 1(d). Densitometry is often used to quantify spots or bands on colorimetrically stained protein gels or on X-ray films used to image chemiluminescent Western blots or radiolabeled Northern or Southern blots.
An overview of technical considerations for Western blotting Image analysis software typically contain tools that simplify the molecular weight calculations and only require you to identify what standard was loaded on the gel. Next, a reference lane is selected on the blot. Any blots not stained with Ponceau S were rehydrated and put through the same destaining washes as blots that were stained. Federal government websites often end in .gov or .mil. The site is secure. ISEQ00010, EMD Millipore) in 1 NuPAGE Transfer Buffer (Cat. 10L of each sample (2g of total protein for samples treated as unknowns, and between 0.125 and 4g of total protein for standards) was combined with 6.25L of Amersham QuickStain labelling buffer, 6.25L of Bolt 4 LDS Buffer, and 1L of undiluted Cy5 stain from the Amersham QuickStain kit. This selection was changed to prevent counting (as background) the signal from overlapping between neighbouring lanes as well as any nontarget bands 5-10kDa away from the target protein molecular weight as these were likely to interfere with proper background subtraction if unaccounted for. Proteins were then transferred onto Optitran BA-S 85 Reinforced Nitrocellulose (Cat. 2023 May 24;11(20):4377-4388. doi: 10.1039/d3tb00288h. SDS-PAGE and transfer steps were performed in an XCell SureLock Mini Gel Tank (Thermo Fisher Scientific) on a Power Zoom-dual power pack or Power Ease 500 power pack (Thermo Fisher Scientific). After loading the gels, proteins were separated at 165V and 125mA for 45min in 1 Bolt MOPS SDS Running Buffer (Cat. These changes in abundance may be due to in vivo biological differences [18], may be induced by the experimental treatments under investigation [7, 41], or may be related to the in vitro biology of experimental models [43]. Townsend M, Fowler B, Aulakh GK, Singh B. Unless total protein labelling has been shown to interfere with immunodetection, there are two important reasons why every Western blot membrane should be labelled for total protein and these data presented even when loading control protein(s) are also detected using antibodies: It is also important to recognise that labelling all proteins in a sample does not discriminate between the sources of protein when more than one biological source is present [3]. The protein concentration in pooled samples was reestimated using the appropriate quantification kit listed above for 2D or SDS buffer. We confirm that ghosting artefacts associated with overabundance of proteins of interest in Western blots can skew findings. No. Tips, Techniques, and Technologies from the Western Blotting Experts at Bio-Rad Laboratories. In either case, any variation in the signal of the reference loading control indicates experimental variation rather than a difference in expression of the target protein. B000102, Thermo Fisher Scientific), and then proteins were transferred to Odyssey nitrocellulose membranes (Cat. 2D samples containing recombinant proteins shown in Figure 4 were run on the same type of gels and transferred to the same membrane type using identical conditions. 2014;2014:361590. doi: 10.1155/2014/361590. Reassessing Western blotting. Butler TAJ, Paul JW, Chan EC, Smith R, Tolosa JM. Be careful to capture the entire lane, but avoid including neighboring lanes. This may provide a better fit for gels with non-uniform acrylamide or gels with some bands in the stacking gel.. data are approximated by proportional linear models), the investigator must demonstrate that normalisation error cannot explain their findings and the data used to reach this conclusion should be shown. Multiple explanations for this type of saturation have been suggested, including that it may be due to: Regardless of the cause of saturation, our work and the work of others [221, 25] indicate that it should not be assumed that O.D. [Google Scholar] Georgopoulos NT, et al . Dittmer A., Dittmer J. Beta-actin is not a reliable loading control in Western blot analysis. All SDS-PAGE and Western blot transfer of these protein samples were performed in Bolt mini-gel tanks (Thermo Fisher Scientific) using PowerEase 90W power packs (Thermo Fisher Scientific). The design of a quantitative western blot experiment. eCollection 2022. data obtained from total protein labelling often remain linear at lysate loads greater than 5-10 g of protein [2, 79, 12, 15, 17, 18, 20, 25, 39]. How densitometry applies to molecular biology. A subsequent total protein quantification assay (2-D Quant Kit run according to manufacturer's instructions) confirmed that the protein content was still approximately 2mg/mL. Gassmann M, et al. Baker M. Reproducibility crisis: blame it on the antibodies. O'Brien M., Carbin S., Morrison J. J., Smith T. J. This approach utilizes a two-step procedure where each lane of a blot is identified in the image, and then individual bands are identified inside each lane. data with loading control protein O.D. Read a summary about a complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting. To ensure that the entire band was captured, the values in the lookup table were adjusted to increase contrast. In the linear region of detection, O.D. It is common to test multiple potential housekeeping proteins for a given experimental procedure. No.
Quantifying Western blots: pitfalls of densitometry - PubMed The https:// ensures that you are connecting to the Here we provide data that define what should be considered, what avoided and what documented when quantifying Western blots. Western blotting experiments were conducted at room temperature using 1h incubations for membrane blocking, 2h incubations for primary antibodies, and 1h incubations for secondary antibodies. 2019 Jan 21;2019:5214821. doi: 10.1155/2019/5214821. An official website of the United States government. While uncertain antibody specificity threatens the validity of any Western blot assay [2, 3, 20, 3437], a Western blot performed using an antibody that does not recognise the correct target can only be fixed by replacing it with one that does.
Western Blot Detection - Azure Biosystems A., Veiras L. C., Minas J. N., Ralph D. L. Considerations when quantitating protein abundance by immunoblot. To control for membrane-specific effects, it is important that the dilution series of the calibrator is included on each membrane that contains samples being compared [2, 4, 5, 21, 25], and O.D. 23227, Thermo Fisher Scientific). As a result, they exhibit good linearity in the common loading range of 1050 g of cell lysate. Measuring the linear dynamic range of a CCD camera-based imager. All SDS samples were run on 10-well 1 mm thick 4-12% Bolt Bis-Tris Plus gels (Cat. All densitometry data and representative images of Western blots and membranes labelled with total protein stains that were used to support the findings of this study are included within the supplementary materials. data for recombinant ENPP1 measured in the standards fitted a proportional linear equation (Figure 5) (equation y = 702615x, relative R2 = 0.993). When we normalised one of these proteins against another protein (Figures 3(a), 3(b), and 3(c)) or a protein against the Ponceau S stain (Figures 3(d), 3(e), and 3(f)), the normalised ratio was not constant, and it systematically differed across each dilution series in a manner that was specific to the proteins under investigation. data do not directly correspond to protein abundance, and if this is not accounted for, any estimates of protein abundance differences between samples will be incorrect [2, 5]. Here we provide data that define what should be considered, what avoided and what documented when quantifying Western blots. Before . It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex protein mixtures. You need to strip the blots for each and incubate the blots with loading control (B-actin). Quantitating a western blot refers to the measurement of the signal emitted by your protein band(s) of interest. The ImageJ ecosystem: An open platform for biomedical image analysis. It constitutes a multistep method that allows the detection and/or quantification of proteins from simple to complex protein mixtures. Many blot analysis software packages allow you to estimate the molecular weight of your target protein. Stock aliquots of pooled myometrial tissue homogenates were defrosted on ice, warmed to room temperature, and then vortexed to ensure complete suspension of proteins. Explore the features and benefits of the new high-end imaging system for the best fluorescence and chemiluminescence detection. 1996 May 15;237(1):123-8. doi: 10.1006/abio.1996.0209. Pooled pregnant human myometrial tissue homogenates extracted in 2D lysis buffer were run in 2-fold serial dilutions from 40g to 40ng of total protein lysate per lane. Motulsky H. J. Linear proportionality should not be assumed even when based on similar studies, as differences in methodology that appear trivial affect the linearity of O.D. The .gov means its official. Janes K. A. Total protein analysis as a reliable loading control for quantitative fluorescent Western blotting.
Image Analysis and Quantitation for Western Blotting | Bio-Rad Here we use Image Lab Software as an example, but most software packages have similar tools. For the most accurate results, it is best to load standards in both the first and last lanes., Define the lanes on the gel, making sure to set the top of the lanes to be either the bottom of the wells or the top of the resolving gel. Each pixel in these digital images is assigned an intensity value that is related to the number of photons detected by the corresponding pixel in the sensor until it reaches saturation [1]. SYPRO Ruby, Ponceau S, or Bio-Rads Stain-Free technology). Pregnant human myometrial tissue homogenates extracted in 2D lysis buffer were run in independent dilutions at 40, 20, 10, 5, and 2.5g of total protein lysate. Expert Rev Proteomics. A linear dynamic range that extends from high to low signal intensities supports accurate quantitation across an equally broad range of protein concentrations (Fig 1). Campbell J. H., Kocher O., Skalli O., Gabbiani G., Campbell G. R. Cytodifferentiation and expression of alpha-smooth muscle actin mRNA and protein during primary culture of aortic smooth muscle cells, Correlation with cell density and proliferative state. TrkC Intracellular Signalling in the Brain Fear Network During the Formation of a Contextual Fear Memory. NP0322, Thermo Fisher Scientific). Each protein was detected with infrared fluorescence (top figure in each panel) and chemiluminescence (bottom figure in each panel). Fernndez-Fernndez and F. Van Breusegem (2022).Detection of Damage-Activated Metacaspase Activity by Western Blot in Plants. data are linear but nonproportional [2, 5]. 4906837001, Roche) were used in both lysis buffers. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. These two objectives are the driving force behind every decision we make, from developing innovative ideas to building global solutions that help solve our customers' greatest challenges.Connect with Bio-Rad Online:Website: http://www.bio-rad.com/ LinkedIn: https://www.linkedin.com/company/bio-rad Facebook: https://www.facebook.com/biorad/ Twitter: https://twitter.com/BioRadLifeSci Instagram: @BioRadLabsSnapchat: @BioRadLabs Mathematically, any lane can be used as the reference, as the normalization factor will be applied to all other lanes as a ratio relative to the reference lane., Then determine the normalization factor for each lane by deriving the ratio of the signals of the housekeeping protein in each lane, using the reference lane as the numerator. Any other bands visible at different molecular weights were excluded from analyses. When using infrared detection, we performed all steps from the secondary antibody incubation in the dark. O.D. An official website of the United States government. data were unaffected by saturation since they fit linear models. iBright . Discover the advantages that digital imaging has over film. Different common densitometry procedures applied to the identical Western blot revealed p-values of these correlations ranging from 0.000013 to 0.76 reflecting the necessity of a scientifically sound basis for densitometry of Western blots. If these normalised data (ENPP1:Ponceau S or Fam3a:Ponceau S) were used to compare ENPP1 or Fam3a protein levels between these samples, the estimated differences in protein abundance would be incorrect. No. WBLUCO100, EMD Millipore) for 1 minute at room temperature. Electrophoretic gels such as Western blots need frequently to be quantified in order to translate biochemical results into statistical values . data were calculated from band area and background-subtracted intensity for (b) nitrocellulose membranes detected by chemiluminescence, (c) nitrocellulose membranes detected by infrared fluorescence, (d) PVDF membranes detected by chemiluminescence, and (e) PVDF membranes detected by infrared fluorescence. data deviate from proportional linear models, the reliability of normalisation performed by dividing target protein O.D. The absolute amounts of recombinant ENPP1 and Fam3a protein analysed (12.5ng in one sample vs. 50ng in the other) were calculated to be within the linear range of detection, and we included a standard curve covering the range of 3.25100ng of recombinant ENPP1 and Fam3a spiked into myometrial lysate. No. 1) Open Western scan in Image. J Mater Chem B. Detect bands in the standards and sample lanes., In the software, enter the type of standard used (or enter the molecular weight data for standards not listed in your software) and instruct the software which lanes are to be used as standard lanes., The software will automatically plot the molecular weight as a function of Rf and interpolate the molecular weights of your proteins of interest., In Image Lab, other regression methods such as point-to-point can also be used. At Bio-Rad, we are united behind this effort. Non-detector saturated images are used for quantitation, and protein/PTM abundance is most often measured using an optical density (O.D.)