Azure Biosystems
Total protein normalization (TPN) is a laboratory method used to resolve the native difficulties with linearity in the immunodetection of target and control proteins. Total protein normalization is the new standard required by journals for Western blot quantification.
Immunodetection is a technique that uses antibodies to identify specific proteins that have been blotted onto membranes. It can be used for a variety of techniques, like Western blotting and ELISA.
Normalizing a Western blot is crucial for accurate and reliable results in laboratory research. It accounts for variations in total protein levels due to experimental error, minimizing their impact on the final outcome. Normalizing increases accuracy, consistency, and the validity of results. You should perform proper normalization to enhance scientific rigor and contribute to knowledge advancement.
There are two ways to normalize your Western blot: single protein detection and total protein normalization. The single protein detection method uses housekeeping proteins (HKPs) and is often done by comparing the signal for the target protein to the housekeeping protein.
A housekeeping protein is thought to be consistently expressed across different sample types. Its expression is believed to be unaffected by whatever intervention was carried out to produce the samples being compared.
Western blotting will tell you if your protein of interest is present in a sample. To determine if the amount of target protein is different between two or more samples, quantitative blotting is required (Table 1).
Table 1. Two factors of quantitative Western blotting
| Quantitative Western blotting for TPN involves |
|---|
| Ensuring measurements of the protein of interest are within the linear range of the detection system used, and |
| Normalizing the measurements of the protein of interest to control for differences in the amount of sample loaded and/or any differences in transfer efficiency during the blotting protocol. |
HKPs are widely used as an internal reference for Western blot normalization. They are often highly expressed, lose proportionality at high protein loads, and only represent one portion of your whole sample. For this reason, using HKPs is an unreliable method of calculating normalization ratios.
Experimental conditions may change the expression of some commonly used housekeeping proteins. The expression level of the HPK must be similar to that of the protein of interest, otherwise one of the two proteins may not be within the linear range of detection. If the signal for the HPK is saturated under conditions needed to detect the target protein, it is not useful for normalization.
Fortunately, TPN can be used to measure the total protein abundance of each sample . This approach involves either
TPN provides a valuable opportunity for accurately evaluating protein abundance and can be tailored to meet specific research needs.
TPN avoids the pitfalls associated with housekeeping protein normalization. In TPN, the signal for the protein of interest is normalized to the total amount of protein loaded in the lane. TPN provides a larger useful dynamic range, is not dependent on the expression of a housekeeping protein being unaffected by experimental manipulations, and can provide additional information about the quality of electrophoresis and Western blotting. It also takes into account:
Fluorescent total protein stains are used for normalizing with TPN; they are easy to integrate into your existing Western blotting workflow. During Western blotting, after transfer step, stain the blot with either a fluorescent or colorimetric stain before blocking and antibody detection of the target protein. The normalized blots can then be imaged on both camera- and laser-based scanning instruments with a green or Safe-Dyes channel.
There are several stains are compatible with downstream detection, including AzureRed and TotalStainQ. For example, AzureRed is a total protein stain that may be imaged with a 520 nm laser. One to three proteins may also be detected using other fluorescent channels (Figure 1).
TotalStainQ is another fluorescent total protein stain that is compatible with downstream chemiluminescent or NIR detection (Figure 2). Using TotalStainQ doesn’t require a destaining step. If you’re using other total protein stains, your Western blot may need to be imaged and destained before the blocking and antibody detection steps.
Combine TotalStain Q with your downstream near-infrared or chemiluminescent imaging protocol for quantitative analysis. It accurately accounts for lane-to-lane variations inherent in Western blotting. With linearity up to 50 μg of lysate, it is ideal for the normalization of lowly expressed proteins that require high sample loads.
For ease of use, Azure offers two kits for normalizing Western blots for use with either PVDF or nitrocellulose membranes. Request a Sample here >>
Watch this quick, 10-minute on-demand webinar using TotalStain Q for quantitative Western blotting results.
Azure Biosystems offers a full range of imaging systems, which include several models that allow target protein detection to be multiplexed with TPN– with no need for dedicated precast gels or laborious stripping and re-probing. Instead, treat your blots with a fluorescent protein stain like TotalStain Q, between protein transfer and blocking, and process them as you would normally.
Compare imaging systems capable of imaging TPN here.
For TPN with NIR Western blot detection, the Azure 500Q and the new Sapphire FL both provide detection of target analytes in the 700nm and 800nm channels, with a third channel used to measure TotalStain Q. Read more >>
The common practice for getting quantitative data from Western blots has been to normalize your band of interest to the signal from a housekeeping protein or to normalize to total protein. Read more about how to normalize your blot to total protein
Total protein normalization uses the entire protein content of each sample for normalization instead of relying on only a single housekeeping protein. Check out this example of total protein staining that shows detection of total protein in six identical samples of HeLa lysate before immunodetecting pSTAT3.
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