Dealing with large sample sizes: comparison of a new one spot dot blot method to western blot
- Background: Western blot is the gold standard method to determine individual protein expression levels. However, western blot is technically difficult to perform in large sample sizes because it is a time consuming and labor intensive process. Dot blot is often used instead when dealing with large sample sizes, but the main disadvantage of the existing dot blot techniques, is the absence of signal normalization to a housekeeping protein. Methods: In this study we established a one dot two development signals (ODTDS) dot blot method employing two different signal development systems. The first signal from the protein of interest was detected by horseradish peroxidase (HRP). The second signal, detecting the housekeeping protein, was obtained by using alkaline phosphatase (AP). Results: Inter-assay results variations within ODTDS dot blot and western blot and intra-assay variations between both methods were low (1.04 - 5.71%) as assessed by coefficient of variation. Conclusions: ODTDS dot blot technique can be used instead of westernBackground: Western blot is the gold standard method to determine individual protein expression levels. However, western blot is technically difficult to perform in large sample sizes because it is a time consuming and labor intensive process. Dot blot is often used instead when dealing with large sample sizes, but the main disadvantage of the existing dot blot techniques, is the absence of signal normalization to a housekeeping protein. Methods: In this study we established a one dot two development signals (ODTDS) dot blot method employing two different signal development systems. The first signal from the protein of interest was detected by horseradish peroxidase (HRP). The second signal, detecting the housekeeping protein, was obtained by using alkaline phosphatase (AP). Results: Inter-assay results variations within ODTDS dot blot and western blot and intra-assay variations between both methods were low (1.04 - 5.71%) as assessed by coefficient of variation. Conclusions: ODTDS dot blot technique can be used instead of western blot when dealing with large sample sizes without a reduction in results accuracy.…
Author details: | Sulistyo Emantoko Dwi Putra, Oleg TsuprykovGND, Karoline Von WebskyORCiDGND, Teresa Ritter, Christoph ReichetzederORCiDGND, Berthold HocherORCiDGND |
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DOI: | https://doi.org/10.7754/Clin.Lab.2014.140317 |
ISSN: | 1433-6510 |
Pubmed ID: | https://pubmed.ncbi.nlm.nih.gov/25648029 |
Title of parent work (English): | Clinical laboratory : the peer reviewed journal for clinical laboratories and laboratories related to blood transfusion |
Publisher: | Clin Lab Publ., Verl. Klinisches Labor |
Place of publishing: | Heidelberg |
Publication type: | Article |
Language: | English |
Year of first publication: | 2014 |
Publication year: | 2014 |
Release date: | 2017/03/27 |
Tag: | comparison; large sample size studies; one dot two development signals (ODTDS) dot blot; protein quantification; western blot |
Volume: | 60 |
Issue: | 11 |
Number of pages: | 7 |
First page: | 1871 |
Last Page: | 1877 |
Organizational units: | Mathematisch-Naturwissenschaftliche Fakultät / Institut für Ernährungswissenschaft |
Peer review: | Referiert |