Premium
Considerations for Western Blot Normalization Techniques
Author(s) -
Oh Kenneth,
Dreskin Elizabeth Jordan,
Liu Ning,
Hammond Matthew,
Kollmann Franziska,
Posch Anton
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.802.15
Subject(s) - normalization (sociology) , housekeeping gene , stain , database normalization , blot , western blot , housekeeping , chemistry , staining , computational biology , computer science , biology , pattern recognition (psychology) , artificial intelligence , biochemistry , gene expression , genetics , anthropology , gene , sociology
Western blotting is a well‐established method to specifically measure the relative quantities of individual proteins in complex biological samples. However, data quality may be affected by technical and biological variation. The application of data normalization strategies is strongly recommended to compensate for these non‐sample related variations in signal intensity. Here, we compare the power of two different data normalization strategies, namely housekeeping protein (HKP) and total protein (TP) normalization based on Stain‐free technology to offer an orientation in selecting the appropriate data normalization tool. In brief, HeLa cell lysates were separated with SDS‐PAGE Stain‐free gels and blotted onto a PVDF membrane. Stain‐free imaging after activation with UV was applied to obtain a total protein blot signal. The blot was then treated to the appropriate immuno‐probing steps and the intensity of the target protein ATG7 was recorded by chemiluminescence. In order to illustrate the differences of the two techniques, the membrane was subsequently immuno‐probed for GAPDH as a typical example for housekeeping protein data normalization. Both data sets were used for data normalization of the target protein ATG7. Our results revealed that total protein staining through Stain‐free technology shows less technical variation than HKP recording as represented by GADPH. Additionally, the use of housekeeping proteins as data normalization tool is restricted in its general applicability and may not be compatible with the strict publishing journal guidelines. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .