In biological data analysis, normalization is an important procedure used to remove both biological and non-biological sources 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. 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. Although Western blot normalization is heading toward total protein normalization instead of using housekeeping proteins, the method is still relevant to genetic studies.
Video Western blot normalization
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. 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. 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. It is critical to carefully validate any housekeeping protein chosen as a normalization standard against the sample type and experimental condition. Although normalization reagents (such as antibodies against key proteins) are common and effective, HKP expression can be impacted by experimental conditions. As a result, normalization control must be validated.
The detection method in this section is antibody-based and 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. Many housekeeping proteins are expressed at high levels, and are 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.
While antibodies are commercially available, validation techniques may not be consistent and poorly characterized antibodies may yield nonspecific results with low reproducibility.
Other complications with normalization techniques include efficiency and reagent properties. 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. The loading control protein must also 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.
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. A known quantity of protein is added which can be controlled ensuring that the detection is within the linear range of the antibody. An advantage to this method is that there are more proteins to choose from as controls, than HKP. However, expression of the protein of interest is only compared to one other protein. 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
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. TPN was first introduced in 1995, but became widely adopted for western blot normalization around 2008. 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. Alternatively, total protein normalization has been proposed as an improved, more appropriate strategy for sample normalization. TPN involves incubating the gel or membrane with a total protein stain, either before or after detection with antibodies. A more efficient stain-free method has also recently become available.
TPN is not dependent on a single loading control, increasing accuracy as normalization is done against many proteins. There is also no need to carry out extensive validation of controls or strip/reprobe blots for detection of housekeeping proteins. These factors can improve the precision (down to 0.1 µg of total protein per lane), cost-effectiveness, and reliability of western blotting data.
Even though precision is high with TPN, the fluorescent stains and stain-free gels require equipment to be visualized, and staining may not be evenly done across the blot. The edges can get more intensely stained than the center of the blot.
Maps Western blot normalization
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. 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.
Gel-staining techniques
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 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 ?g is reported for Ponceau S with poor reproducibility due to its highly time-dependent staining intensity and low signal-to-noise ratio.
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. 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 ?g of protein per lane.
Post antibody stains
Amido black is a typically used permanent post-antibody anionic stain with higher sensitivity than Ponceau S. 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. 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.
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. Consequently, it is becoming more popular, with an exponential growth in the number of publications using this technique. Gels developed with stain-free technology are compatible with standard SDS-PAGE buffers. Stain-free technology can also visualize proteins in the gel after electrophoresis or on the blot after transfer. Modifications to the proteins themselves are minimal and do not affect protein transfer or downstream antibody binding in western blotting. Furthermore, the observed intensity of the bands does not depend on the duration of staining/destaining and does not decrease with time.
The linear range for stain-free normalization 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. 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.
Despite its sensitivity, stain-free technology cannot detect proteins that do not contain tryptophans, and it is recommended that a protein contains at least two tryptophans to be readily detected.
Journal guidelines
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. 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.
References
External links
- V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting
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