PMID- 36645159
OWN - NLM
STAT- MEDLINE
DCOM- 20230512
LR  - 20230521
IS  - 1522-2683 (Electronic)
IS  - 0173-0835 (Linking)
VI  - 44
IP  - 9-10
DP  - 2023 May
TI  - Western blot normalization: Time to choose a proper loading control seriously.
PG  - 854-863
LID - 10.1002/elps.202200222 [doi]
AB  - Recent research has questioned the validity of housekeeping proteins in Western 
      blot. Our present study proposed new ideas for Western blot normalization that 
      improved the reproducibility of scientific research. We used the Gene Expression 
      Omnibus (GEO) database and the web tool GEO2R to exclude unstable housekeeping 
      genes quickly. In ischemic heart tissues, actin and tubulin changed 
      significantly, whereas no statistically significant changes were observed in the 
      expression of genes relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). 
      Besides, the reliability of GAPDH was further examined by Western blot. 
      Additionally, unstable housekeeping genes were found in other animal models of 
      cardiovascular medicine. We also found that sodium dodecyl sulfate and 
      temperature significantly impacted the results of Ponceau S staining. Membranes 
      stained with Ponceau S after immunodetection could avoid this interference, and 
      the coefficients of variation for post-immunodetection staining are lower than 
      those produced by GAPDH immunodetection. Overall, we described a new use of 
      differential gene expression analysis and proposed a modified Ponceau S staining 
      method, which provided researchers with a proper loading control for Western blot 
      and hence could improve reproducibility in research.
CI  - (c) 2023 Wiley-VCH GmbH.
FAU - Wang, Qinhong
AU  - Wang Q
AUID- ORCID: 0000-0001-7553-2616
AD  - Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese 
      Ministry of Education, Chinese National Health Commission and Chinese Academy of 
      Medical Sciences, State and Shandong Province Joint Key Laboratory of 
      Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital, 
      Cheeloo College of Medicine, Shandong University, Jinan, Shandong, P. R. China.
FAU - Han, Wenqiang
AU  - Han W
AD  - Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese 
      Ministry of Education, Chinese National Health Commission and Chinese Academy of 
      Medical Sciences, State and Shandong Province Joint Key Laboratory of 
      Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital, 
      Cheeloo College of Medicine, Shandong University, Jinan, Shandong, P. R. China.
FAU - Ma, Chuanzhen
AU  - Ma C
AD  - Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese 
      Ministry of Education, Chinese National Health Commission and Chinese Academy of 
      Medical Sciences, State and Shandong Province Joint Key Laboratory of 
      Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital, 
      Cheeloo College of Medicine, Shandong University, Jinan, Shandong, P. R. China.
FAU - Wang, Tianyu
AU  - Wang T
AD  - Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese 
      Ministry of Education, Chinese National Health Commission and Chinese Academy of 
      Medical Sciences, State and Shandong Province Joint Key Laboratory of 
      Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital, 
      Cheeloo College of Medicine, Shandong University, Jinan, Shandong, P. R. China.
FAU - Zhong, Jingquan
AU  - Zhong J
AD  - Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese 
      Ministry of Education, Chinese National Health Commission and Chinese Academy of 
      Medical Sciences, State and Shandong Province Joint Key Laboratory of 
      Translational Cardiovascular Medicine, Department of Cardiology, Qilu Hospital, 
      Cheeloo College of Medicine, Shandong University, Jinan, Shandong, P. R. China.
AD  - Department of Cardiology, Qilu Hospital (Qingdao), Cheeloo College of Medicine, 
      Shandong University, Qingdao, Shandong, P. R. China.
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20230131
PL  - Germany
TA  - Electrophoresis
JT  - Electrophoresis
JID - 8204476
RN  - 0 (Actins)
RN  - EC 1.2.1.- (Glyceraldehyde-3-Phosphate Dehydrogenases)
RN  - T1C2UGS05F (ponceau S)
SB  - IM
MH  - Animals
MH  - Reproducibility of Results
MH  - *Actins/analysis
MH  - *Glyceraldehyde-3-Phosphate Dehydrogenases/genetics/analysis/metabolism
MH  - Staining and Labeling
MH  - Blotting, Western
OTO - NOTNLM
OT  - GEO database
OT  - Ponceau S staining
OT  - Western blot
OT  - loading control
OT  - normalization
EDAT- 2023/01/17 06:00
MHDA- 2023/05/12 07:06
CRDT- 2023/01/16 07:13
PHST- 2022/12/28 00:00 [revised]
PHST- 2022/09/11 00:00 [received]
PHST- 2023/01/03 00:00 [accepted]
PHST- 2023/05/12 07:06 [medline]
PHST- 2023/01/17 06:00 [pubmed]
PHST- 2023/01/16 07:13 [entrez]
AID - 10.1002/elps.202200222 [doi]
PST - ppublish
SO  - Electrophoresis. 2023 May;44(9-10):854-863. doi: 10.1002/elps.202200222. Epub 
      2023 Jan 31.