Western blot normalization

Introduction to normalization

In biological data analysis, normalization is an important procedure used to remove both biological and non-biological sources[1][2] of experimental variability. For accurate, reproducible quantitative analysis of proteins via western blotting, normalization accounts for differences arising from cell culture conditions, inconsistent sample preparation, uneven protein transfer and unequal sample loading across gel lanes.[3] Traditionally for western blots, normalization involves comparing the relative abundance of the protein of interest to that of an unrelated control protein such as a housekeeping protein, to ensure that visually assessed changes in protein levels represent true biological variation and not experimental artifacts.[3][4]

Normalization Methods

Normalization with single proteins: Housekeeping proteins (HKPs) and spike-in controls

Housekeeping proteins as loading controls

The normalization control is a protein that, ideally, is expressed constantly at the same level across different experimental conditions. As such, housekeeping genes and proteins are often used as internal normalization standards in quantitative PCR (qPCR) and western blots.[4][5][6] These proteins were designated as housekeeping because they are essential for the maintenance of basic cellular functions and are universally expressed in all cells of an organism. Some examples of classically used housekeeping proteins include ß-Actin, GAPDH, HPRT1, and RPLP1.[3][5] Until recently, these proteins were considered to be expressed at a consistent, stable level under normal or pathophysiological conditions in any cell or tissue type. However, recent studies have shown that expression of housekeeping proteins can change across different cell types and biological conditions.[3][7][8][9] It is critical to carefully validate any housekeeping protein chosen as a normalization standard against the sample type and experimental condition.

Advantages of using HKPs
  1. Antibodies against key proteins are readily available from many commercial suppliers and are cost-effective.[10]
  2. No new skills or equipment required.
Disadvantages of using HKPs
  1. HKP expression can be impacted by experimental conditions. Therefore, normalization control must be validated.[7][8][9]
  2. The detection method used here is antibody-based and therefore requires a linear relationship between signal intensity and the sample mass or volume to be confirmed for every antigen. Both the target protein and the normalization control should be within the dynamic range of detection.[3] Many housekeeping proteins are expressed at high levels, and only appropriate for use with highly expressed target proteins. Lower expressing proteins are difficult to detect on the same blot. This is further complicated by the fact that some detection methods, in particular, enhanced chemiluminescence using x-ray film, have a very limited linear range.[11]
  3. While antibodies are commercially available, validation techniques may not be consistent and poorly characterized antibodies may yield nonspecific results with low reproducibility.[10][12][13]
  4. Membranes need to be stripped and re-probed when detecting multiple protein targets on the same blot. Ineffective stripping could result in signal contamination. It is time-consuming and can increase the number of reagents required. Additionally, membrane stripping cannot be performed indefinitely. Usually, only three stripping incubations are recommended per membrane.[5]
  5. The loading control protein must be considerably different in molecular weight than the target protein in cases where fluorescent detection is not utilized. Care must be taken for the two proteins to be adequately separated by gel electrophoresis for accurate analysis.[3]
  6. Expression of the target protein is only compared to one other protein.

Exogenous spike-in controls

A pure protein added externally can be used as a normalization control if it is spiked into each of the samples at a known concentration.

Advantages of using spike-in controls
  1. A known quantity of protein is added which can be controlled ensuring that the detection is within the linear range of the antibody.
  2. Control protein is consistent across all experimental conditions.
  3. There are more proteins to choose from as controls, than HKP.[14]
Disadvantages
  1. Expression of the protein of interest is only compared to one other protein.
  2. Spike-in proteins can control only for certain steps in the western blotting process, depending upon when they are introduced. When introduced after sample extractions from cells or tissues, the spike protein only controls for differences in sample loading and transfer but not any experimental error in sample preparation methods.

Normalization with total protein

What is TPN? Why and when was it introduced?

In total protein normalization (TPN), the abundance of the target protein is normalized to the total amount of protein in each lane, removing variations associated with normalization against a single protein.[15][16] TPN was first introduced in 1995,[17] but became widely adopted for western blot normalization around 2008.[15] Re-evaluation of the housekeeping protein normalization technique led to the finding that HKP loading controls may not be as accurate as initially thought since there are experimental conditions that can affect their expression.[4][5] Alternatively, total protein normalization has been proposed as an improved, more appropriate strategy for sample normalization.[18] TPN involves incubating the gel or membrane with a total protein stain, either before or after detection with antibodies.[15][16] A more efficient stain-free method has also recently become available.[19][20]

Advantages
  1. TPN is not dependent on a single loading control. More accurate because normalization is done against many proteins.[21]
  2. No need to carry out extensive validation of controls.[11]
  3. Eliminates the need to strip and reprobe blots for detection of housekeeping proteins to normalize protein levels.[5]
  4. Saves time and improves the precision and reliability of western blotting data.
  5. Acceptable in most journals as a valid normalization method for western blot.[11]
  6. A large range of sensitivities (down to low femtogram levels) can be accessed.
  7. TPN is more compatible with detecting proteins of lower abundance.[3]
Disadvantages
  1. Fluorescent stains and stain-free gels require equipment to be visualized.[21]
  2. Staining may not be evenly done across the blot. The edges can get more intensely stained than the center of the blot.[3]

Procedure

Theory

Normalization is performed by measuring total protein directly on the gel or membrane that is used for western blotting. The stained gel or blot is imaged, and normally a rectangle is drawn to include all proteins in a lane. The signal intensity inside this rectangle is used to represent that sample’s total protein content in normalization calculations.[3] The signal intensity of the protein of interest is normalized to this value. When using protein stains, the membrane may be incubated with the chosen stain before or after immunodetection, depending on the type of stain.[21]

Gel-staining Techniques

Pre-antibody stains

Anionic dyes such as Ponceau S and Coomassie Brilliant Blue,[22][23] and fluorescent dyes like Sypro Ruby and Deep Purple are used before the addition of antibodies. They do not affect the downstream immunodetection.

Ponceau S is a negatively charged reversible dye that stains proteins a reddish pink color and is removed easily by washing in water. The intensity of Ponceau S staining decreases quickly over time, so documentation should be conducted rapidly. 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.[24][25]

Sypro Ruby and other fluorescent dyes have a broad linear range and higher sensitivity than anionic dyes. They are permanent photostable stains that can be visualized with a standard UV or blue-light transilluminator or a laser scan.[3][25] Membranes can then be documented either on film or digitally using a CCD camera. Sypro Ruby blot staining is time-intensive and tends to saturate above 50 μg of protein per lane.[25]

Post antibody stains

Amido black is a typically used permanent post-antibody anionic stain with higher sensitivity than Ponceau S.[3] It must be used after immunodetection. Although not as sensitive as fluorescent dyes, it does not require special equipment for visualization and thus is more economical. Amido black produces bright black bands as the name suggests.

Stain-Free Technology

Stain-Free technology employs an in-gel chemistry for staining. Pre-cast gels are commercially available as well as casting kits and hand-cast gels.[26][27] The gel formulation consists of a trihalo compound that catalyzes a covalent reaction in the presence of tryptophan residues, when exposed to ultraviolet (UV) irradiation. The resulting ‘‘activated’’ protein fluoresces under UV excitation and can be readily detected by fluorescent imaging systems, either within the gel or after transfer to a blotting membrane.[25][26][27]

Because stain-free technology does not require staining and destaining steps, it can be significantly faster than methods that utilize Ponceau S or Sypro Ruby stains.[28] Consequently, it is becoming more popular, with an exponential growth in the number of publications using this technique.[18] Gels developed with stain-free technology are compatible with standard SDS-PAGE buffers.[28]

Advantages

  1. Does not require additional staining reagents or steps to visualize total protein
  2. Higher sensitivity than anionic stains.[28]
  3. Can visualize proteins in the gel after electrophoresis or on the blot after transfer.[29]
  4. Modifications to the proteins themselves are minimal and do not affect protein transfer or downstream antibody binding in western blotting.[29]
  5. The observed intensity of the bands does not depend on the duration of staining/destaining and does not decrease with time.[28][29]
  6. Linear range is up to 80 µg protein for 18- well and up to 110 µg per lane for 12- well Criterion mid-size gels. This range works well with typical protein loads in quantitative western blots and enables loading control calculations over a wide protein-loading range.[18][19] In contrast, conventional stains such as Ponceau S and Sypro Ruby show high variability, and poor reproducibility and linearity in the range of 50-140 ug.[27, 31] When using high protein loads, Stain-Free technology has demonstrated higher success.[28][29]

Disadvantages

  1. Requires special imaging equipment for visualization.
  2. Cannot detect proteins that do not contain tryptophans (recommended that a protein contains at least two tryptophans to be readily detected[30]).

Journal guidelines for western blot normalization

Many journals now require strict adherence to the use of internal controls and are mandating the use of imaging techniques that yield linear signal ranges and report a linear dynamic range of the signal.[11][31] Particularly in the Journal of Biological Chemistry (JBC), normalization of signal intensity to total protein loading (determined by staining membranes, or using stain-free technology) is preferred over the use of housekeeping proteins.[29]

Protocols

References

  1. ↑ Transcriptomics in Health and Disease | Geraldo A. Passos | Springer.
  2. ↑ Heber, Steffen; Sick, Beate (2006-09-01). "Quality Assessment of Affymetrix GeneChip Data". OMICS: A Journal of Integrative Biology. 10 (3): 358–368. doi:10.1089/omi.2006.10.358. ISSN 1536-2310.
  3. 1 2 3 4 5 6 7 8 9 10 11 "A defined methodology for reliable quantification of Western blot data.". Mol Biotechnol. 55. 2016-08-23. PMID 23709336.
  4. 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. ISSN 0168-1656. PMID 10617337.
  5. 1 2 3 4 5 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. doi:10.1111/sms.12702. ISSN 1600-0838. PMID 27263489.
  6. ↑ 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. PMID 23454168.
  7. 1 2 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.
  8. 1 2 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. doi:10.1152/physiolgenomics.00025.2005. ISSN 1094-8341.
  9. 1 2 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.
  10. 1 2 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 2605718Freely accessible.
  11. 1 2 3 4 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 4705984Freely accessible.
  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). doi:10.1371/journal.pone.0135392. ISSN 1932-6203. PMC 4545415Freely accessible.
  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. Retrieved 2016-10-01.
  15. 1 2 3 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.
  16. 1 2 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.
  17. ↑ Klein, D.; Kern, R. M.; Sokol, R. Z. (1995-05-01). "A method for quantification and correction of proteins after transfer to immobilization membranes". Biochemistry and Molecular Biology International. 36 (1): 59–66. ISSN 1039-9712. PMID 7545052.
  18. 1 2 3 Simonyi, K and Yadav, G. (09/07/2016). "Trends in Quantitative Western Blotting – the Emergence of Total Protein Normalization". Bioscience Technology. Advantage Media. Check date values in: |date= (help)
  19. 1 2 "Stain-Free Approach for Western Blotting | GEN Magazine Articles | GEN". GEN. Retrieved 2016-10-01.
  20. ↑ Gilda, JenniferE.; Gomes, AldrinV. (2015-01-01). Posch, Anton, ed. Proteomic Profiling. Methods in Molecular Biology. Springer New York. pp. 381–391. doi:10.1007/978-1-4939-2550-6_27. ISBN 9781493925490.
  21. 1 2 3 "Total Protein Normalization for Western Blots - Advansta Inc.". 2014-11-05. Retrieved 2016-10-01.
  22. ↑ 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.
  23. ↑ 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.
  24. ↑ 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. ISSN 1096-0309. PMID 25193447.
  25. 1 2 3 4 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.
  26. 1 2 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.
  27. 1 2 "Stain-Free Technology | Applications & Technologies | Bio-Rad". www.bio-rad.com. Retrieved 2016-10-01.
  28. 1 2 3 4 5 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.
  29. 1 2 3 4 5 Colella, Alex D.; Chegenii, Nusha; Tea, Melinda N.; Gibbins, Ian L.; Williams, Keryn A.; Chataway, Tim K. (2012-11-15). "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.
  30. ↑ "Total Protein Normalization for Western Blots - Advansta Inc.". Retrieved 2016-10-01.
  31. ↑ "The Journal of Biological Chemistry: Instructions for Authors". www.jbc.org. Retrieved 2016-10-01.
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