Normalization of Western blot data is an analytical step that is performed to compare the relative abundance of a specific protein across the lanes of a blot or gel under diverse experimental treatments, or across tissues or developmental stages.[1][2] The overall goal of normalization is to minimize effects arising from variations in experimental errors, such as inconsistent sample preparation, unequal sample loading across gel lanes, or uneven protein transfer, which can compromise the conclusions that can be obtained from Western blot data.[1] Currently, there are two methods for normalizing Western blot data: (i) housekeeping protein normalization and (ii) total protein normalization.[1][2][3][4]

Procedure

Normalization occurs directly on either the gel or the blotting membrane. First, the stained gel or blot is imaged, a rectangle is drawn around the target protein in each lane, and the signal intensity inside the rectangle is measured.[1] The signal intensity obtained can then be normalized with respect to the signal intensity of the loading internal control detected on the same gel or blot.[1] When using protein stains, the membrane may be incubated with the chosen stain before or after immunodetection, depending on the type of stain.[5]

Housekeeping protein controls

Housekeeping genes and proteins, including β-Actin, GAPDH, HPRT1, and RPLP1, are often used as internal controls in western blots because they are thought to be expressed constitutively, at the same levels, across experiments.[1][2][6][7] However, recent studies have shown that expression of housekeeping proteins (HKPs) can change across different cell types and biological conditions.[1][8][9][10] Therefore, scientific publishers and funding agencies now require that normalization controls be previously validated for each experiment to ensure reproducibility and accuracy of the results.[8][9][10]

Fluorescent antibodies

When using fluorescent antibodies to image proteins in western blots, normalization requires that the user define the upper and lower limits of quantitation and characterize the linear relationship between signal intensity and the sample mass volume for each antigen.[1] Both the target protein and the normalization control need to fluoresce within the dynamic range of detection.[1] Many HKPs are expressed at high levels and are preferred for use with highly-expressed target proteins.[1] Lower expressing proteins are difficult to detect on the same blot.[1]

Fluorescent antibodies are commercially available, and fully characterized antibodies are recommended to ensure consistency of results.[11][12][13]

When fluorescent detection is not utilized, the loading control protein and the protein of interest must differ considerably in molecular weight so they are adequately separated by gel electrophoresis for accurate analysis.[1]

Membrane stripping

Membranes need to be stripped and re-probed using a new set of detection antibodies when detecting multiple protein targets on the same blot.[6] Ineffective stripping could result in a weak signal from the target protein.[6] To prevent loss of the antigen, only three stripping incubations are recommended per membrane.[6] It could be difficult to completely eliminate signal from highly-abundant proteins, so it is recommended that one detects lowly-expressed proteins first.[6]

Exogenous spike-in controls

Since HKP levels can be inconsistent between tissues, scientists can control for the protein of interest by spiking in a pure, exogenous protein of a known concentration within the linear range of the antibody.[8][9][10] Compared to HKP, a wider variety of proteins are available for spike-in controls.[14]

Total protein normalization

In total protein normalization (TPN), the abundance of the target protein is normalized to the total amount of protein in each lane.[3][4] Because TPN is not dependent on a single loading control, validation of controls and stripping/reprobing of blots for detection of HKPs is not necessary.[6][15] This can improve precision (down to 0.1 µg of total protein per lane), cost-effectiveness, and data reliability.[16]

Fluorescent stains and stain-free gels require special equipment to visualize the proteins on the gel/blot.[5] Stains may not cover the blot evenly; more stain might collect towards the edges of the blot than in the center. Non-uniformity in the image can result in inaccurate normalization.[1]

Pre-antibody stains

Anionic dyes such as Ponceau S and Coomassie brilliant blue, and fluorescent dyes like Sypro Ruby and Deep Purple, are used before antibodies are added because they do not affect downstream immunodetection.[17][18][19][20]

Ponceau S is a negatively charged reversible dye that stains proteins a reddish pink color and is removed easily by washing in water.[21][22] The intensity of Ponceau S staining decreases quickly over time, so documentation should be conducted rapidly.[5] A linear range of up to 140 µg is reported for Ponceau S with poor reproducibility due to its highly time-dependent staining intensity and low signal-to-noise ratio.[21][22]

Fluorescent dyes like Sypro Ruby have a broad linear range and are more sensitive than anionic dyes.[22] They are permanent, photostable stains that can be visualized with a standard UV or blue-light transilluminator or a laser scan.[1][22] Membranes can then be documented either on film or digitally using a charge-coupled device camera.[23] Sypro Ruby blot staining is time-intensive and tends to saturate above 50 μg of protein per lane.[22]

Post-antibody stains

Amido black is a commonly used permanent post-antibody anionic stain that is more sensitive than Ponceau S.[24] This stain is applied after immunodetection.[24]

Stain-free technology

Stain-free technology employs an in-gel chemistry for imaging.[22][25][26] This chemical reaction does not affect protein transfer or downstream antibody binding.[27] Also, it does not involve staining/destaining steps, and the intensity of the bands remain constant over time.[28]

Stain-free technology cannot detect proteins that do not contain tryptophan residues. A minimum of two tryptophans is needed to enable detection.[5] The linear range for stain-free normalization is up to 80 µg of protein per lane for 18-well and up to 100 µg per lane for 12-well Criterion mid-sized gels. This range is compatible with typical protein loads in quantitative western blots and enables loading control calculations over a wide protein-loading range.[29][4] A more efficient stain-free method has also recently become available.[30][31] When using high protein loads, stain-free technology has demonstrated greater success than stains.[29]

References

  1. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Taylor, SC; Berkelman, T; Yadav, G; Hammond, M (2016-08-23). "A defined methodology for reliable quantification of Western blot data". Mol. Biotechnol. 55 (3): 217–26. doi:10.1007/s12033-013-9672-6. PMC 3840294. PMID 23709336.
  2. 1 2 3 Thellin, O.; Zorzi, W.; Lakaye, B.; De Borman, B.; Coumans, B.; Hennen, G.; Grisar, T.; Igout, A.; Heinen, E. (1999-10-08). "Housekeeping genes as internal standards: use and limits". Journal of Biotechnology. 75 (2–3): 291–295. doi:10.1016/s0168-1656(99)00163-7. hdl:2268/3661. ISSN 0168-1656. PMID 10617337.
  3. 1 2 Aldridge, Georgina M.; Podrebarac, David M.; Greenough, William T.; Weiler, Ivan Jeanne (2008-07-30). "The use of total protein stains as loading controls: An alternative to high-abundance single-protein controls in semi-quantitative immunoblotting". Journal of Neuroscience Methods. 172 (2): 250–254. doi:10.1016/j.jneumeth.2008.05.003. PMC 2567873. PMID 18571732.
  4. 1 2 3 Collins, Mahlon A.; An, Jiyan; Peller, Danielle; Bowser, Robert (2015-08-15). "Total protein is an effective loading control for cerebrospinal fluid western blots". Journal of Neuroscience Methods. 251: 72–82. doi:10.1016/j.jneumeth.2015.05.011. PMC 4540354. PMID 26004848.
  5. 1 2 3 4 "Total Protein Normalization for Western Blots - Advansta Inc". 2014-11-05. Retrieved 2016-10-01.
  6. 1 2 3 4 5 6 Bass, J. J.; Wilkinson, D. J.; Rankin, D.; Phillips, B. E.; Szewczyk, N. J.; Smith, K.; Atherton, P. J. (2016-06-05). "An overview of technical considerations for Western blotting applications to physiological research". Scandinavian Journal of Medicine & Science in Sports. 27 (1): 4–25. doi:10.1111/sms.12702. ISSN 1600-0838. PMC 5138151. PMID 27263489.
  7. ↑ Li, Rena; Shen, Yong (2013-04-19). "An old method facing a new challenge: re-visiting housekeeping proteins as internal reference control for neuroscience research". Life Sciences. 92 (13): 747–751. doi:10.1016/j.lfs.2013.02.014. ISSN 1879-0631. PMC 3614345. PMID 23454168.
  8. 1 2 3 Zhu, Jiang; He, Fuhong; Song, Shuhui; Wang, Jing; Yu, Jun (2008-01-01). "How many human genes can be defined as housekeeping with current expression data?". BMC Genomics. 9: 172. doi:10.1186/1471-2164-9-172. ISSN 1471-2164. PMC 2396180. PMID 18416810.
  9. 1 2 3 Barber, Robert D.; Harmer, Dan W.; Coleman, Robert A.; Clark, Brian J. (2005-05-11). "GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues". Physiological Genomics. 21 (3): 389–395. CiteSeerX 10.1.1.459.7039. doi:10.1152/physiolgenomics.00025.2005. ISSN 1094-8341. PMID 15769908.
  10. 1 2 3 Lee, Peter D.; Sladek, Robert; Greenwood, Celia M. T.; Hudson, Thomas J. (2002-02-01). "Control Genes and Variability: Absence of Ubiquitous Reference Transcripts in Diverse Mammalian Expression Studies". Genome Research. 12 (2): 292–297. doi:10.1101/gr.217802. ISSN 1088-9051. PMC 155273. PMID 11827948.
  11. ↑ Couchman, John R. (2016-10-01). "Commercial Antibodies: The Good, Bad, and Really Ugly". Journal of Histochemistry and Cytochemistry. 57 (1): 7–8. doi:10.1369/jhc.2008.952820. ISSN 0022-1554. PMC 2605718. PMID 18854593.
  12. ↑ Gilda, Jennifer E.; Ghosh, Rajeshwary; Cheah, Jenice X.; West, Toni M.; Bodine, Sue C.; Gomes, Aldrin V. (2015-08-19). "Western Blotting Inaccuracies with Unverified Antibodies: Need for a Western Blotting Minimal Reporting Standard (WBMRS)". PLOS ONE. 10 (8): e0135392. Bibcode:2015PLoSO..1035392G. doi:10.1371/journal.pone.0135392. ISSN 1932-6203. PMC 4545415. PMID 26287535.
  13. ↑ "Validating Antibodies: An Urgent Need | The Scientist Magazine®". The Scientist. Retrieved 2016-10-01.
  14. ↑ "6 Changes That'll Make a Big Difference With Your RNA-seq; Part 3 - Cofactor Genomics". cofactorgenomics.com. 19 March 2014. Retrieved 2016-10-01.
  15. ↑ Fosang, Amanda J.; Colbran, Roger J. (2015-12-11). "Transparency Is the Key to Quality". The Journal of Biological Chemistry. 290 (50): 29692–29694. doi:10.1074/jbc.E115.000002. ISSN 0021-9258. PMC 4705984. PMID 26657753.
  16. ↑ Moritz, CP. (2017-09-20). "Tubulin or not tubulin: Heading toward total protein staining as loading control in Western blots" (PDF). Proteomics. 17 (20): 1600189. doi:10.1002/pmic.201600189. PMID 28941183. S2CID 22305461.
  17. ↑ Welinder, Charlotte; Ekblad, Lars (2011-03-04). "Coomassie Staining as Loading Control in Western Blot Analysis". Journal of Proteome Research. 10 (3): 1416–1419. doi:10.1021/pr1011476. ISSN 1535-3893. PMID 21186791.
  18. ↑ Ranganathan, Velvizhi; De, Prabir K. (1996-02-01). "Western Blot of Proteins from Coomassie-Stained Polyacrylamide Gels". Analytical Biochemistry. 234 (1): 102–104. doi:10.1006/abio.1996.0057. PMID 8742090.
  19. ↑ Steinberger, B (2015-05-01). "Evaluation of SYPRO Ruby total protein stain for the normalization of two-dimensional Western blots". Analytical Biochemistry. 476 (1): 17–19. doi:10.1016/j.ab.2015.01.015. PMID 25640586.
  20. ↑ "Total Protein Normalization for Western Blots". Retrieved 2016-10-01.
  21. 1 2 Rivero-Gutiérrez, B.; Anzola, A.; Martínez-Augustin, O.; de Medina, F. Sánchez (2014-12-15). "Stain-free detection as loading control alternative to Ponceau and housekeeping protein immunodetection in Western blotting". Analytical Biochemistry. 467: 1–3. doi:10.1016/j.ab.2014.08.027. hdl:10481/63191. ISSN 1096-0309. PMID 25193447.
  22. 1 2 3 4 5 6 Gürtler, Anne; Kunz, Nancy; Gomolka, Maria; Hornhardt, Sabine; Friedl, Anna A.; McDonald, Kevin; Kohn, Jonathan E.; Posch, Anton (2013-02-15). "Stain-Free technology as a normalization tool in Western blot analysis". Analytical Biochemistry. 433 (2): 105–111. doi:10.1016/j.ab.2012.10.010. PMID 23085117.
  23. ↑ "Fluorescence Imaging: Principles and Methods" (PDF). Retrieved 10 January 2018.
  24. 1 2 Goldman, A; Harper, S; Speicher, DW (2016-11-01). Detection of Proteins on Blot Membranes. Vol. 86. pp. 10.8.1–10.8.11. doi:10.1002/cpps.15. ISBN 9780471140863. PMC 5646381. PMID 27801518. {{cite book}}: |journal= ignored (help)
  25. ↑ Zeitler, Anna F.; Gerrer, Katrin H.; Haas, Rainer; Jiménez-Soto, Luisa F. (2016-07-01). "Optimized semi-quantitative blot analysis in infection assays using the Stain-Free technology". Journal of Microbiological Methods. 126: 38–41. doi:10.1016/j.mimet.2016.04.016. PMID 27150675.
  26. ↑ "Stain-Free Technology | Applications & Technologies | Bio-Rad". www.bio-rad.com. Retrieved 2016-10-01.
  27. ↑ Colella, Alex D.; Chegenii, Nusha; Tea, Melinda N.; Gibbins, Ian L.; Williams, Keryn A.; Chataway, Tim K. (15 November 2012). "Comparison of Stain-Free gels with traditional immunoblot loading control methodology". Analytical Biochemistry. 430 (2): 108–110. doi:10.1016/j.ab.2012.08.015. PMID 22929699.
  28. ↑ Gilda, Jennifer E.; Gomes, Aldrin V. (2013-09-15). "Stain-Free total protein staining is a superior loading control to β-actin for Western blots". Analytical Biochemistry. 440 (2): 186–188. doi:10.1016/j.ab.2013.05.027. PMC 3809032. PMID 23747530.
  29. 1 2 Simonyi, K. & Yadav, G. (7 August 2016). "Trends in Quantitative Western Blotting – the Emergence of Total Protein Normalization". Bioscience Technology. Advantage Media.
  30. ↑ "Stain-Free Approach for Western Blotting | GEN Magazine Articles | GEN". GEN. 15 November 2011. Retrieved 2016-10-01.
  31. ↑ Gilda, JenniferE.; Gomes, AldrinV. (2015-01-01). "Western Blotting Using In-Gel Protein Labeling as a Normalization Control: Stain-Free Technology". In Posch, Anton (ed.). Proteomic Profiling. Methods in Molecular Biology. Vol. 1295. Springer New York. pp. 381–391. doi:10.1007/978-1-4939-2550-6_27. ISBN 9781493925490. PMID 25820735.
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