Bio-Rad eBook - 2019 - 11

Total Protein Normalization

Blot Normalization Methods
Western blot normalization enables you to accurately compare changes in target protein expression by correcting
for inconsistencies in sample preparation, pipetting, and protein transfer. Normalization is required to establish a
correlation between changes in band intensities and biological changes in your samples.
Conventionally, a "housekeeping protein" (HKP) such as β-actin, β-tubulin, or GAPDH is used as a loading control,
with the assumption that the expression levels of these proteins remains constant.
Problems with Housekeeping Proteins
The use of HKPs for western blot normalization is now known to be problematic.1 Reliable assessment of changes
in target protein expression levels requires measurement of both the target protein and the loading control protein
in their linear dynamic ranges for immunodetection. Unfortunately, housekeeping proteins are usually highly
expressed, whereas target proteins are often expressed in low abundance. Thus, to detect the target protein of
interest, large amounts of cell lysate may need to be loaded. This may result in overloading of HKPs, yielding
oversaturated reference bands, out of their linear range. Furthermore, HKP expression levels may not be constant,
but instead vary with different experimental treatments and other factors.
1Gomes A V. Can We Trust Western Blots? Paper presented at:

Experimental Biology; April, 2014; San Diego, CA.

Variation in Housekeeping Protein
Expression Levels
The expression level of housekeeping proteins
can change due to:

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Experimental conditions
Developmental changes
Post-transcriptional regulation
Differences in cell tissue age and type

ACTB
TUBB
GAPDH
HIST1H2BC
NONO
RPS18
Total protein staining

Differences in five candidate housekeeping proteins and total protein staining between tumor and noncancerous tissues in
the validation sample set. Immunodetection measurements of housekeeping protein levels show poor linearity and do not accurately
indicate cell lysate loading levels. Total protein normalization of HKPs yields consistent band intensities. (Hu X, Du S, Yu J, Common
housekeeping proteins are upregulated in colorectal adenocarcinoma and hepatocellular carcinoma, making the total protein a better
"housekeeper." Oncotarget 2016; 7(41): 66679-66688).

Stain-Free Imaging for Total Protein Normalization

The use of total protein measurement for western blot loading controls (total protein normalization; TPN) is a
method devised to eliminate the risk that expression of the loading control is affected by experimental conditions.
Total protein levels can be determined by staining the membrane with total protein stains; however, as discussed
previously, these present their own difficulties.
Stain-Free Imaging with TPN has thus emerged as the best method for quantitative western blotting.

Total Protein Normalization
bio-rad.com/TPN

https://www.bio-rad.com/TPN

Bio-Rad eBook - 2019

Table of Contents for the Digital Edition of Bio-Rad eBook - 2019