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BioTechniques Sep 2023Western blotting (immunoblotting) is a powerful and commonly used technique that is capable of detecting or semiquantifying an individual protein from complex mixtures... (Review)
Review
Western blotting (immunoblotting) is a powerful and commonly used technique that is capable of detecting or semiquantifying an individual protein from complex mixtures of proteins extracted from cells or tissues. The history surrounding the origin of western blotting, the theory behind the western blotting technique, a comprehensive protocol and the uses of western blotting are presented. Lesser known and significant problems in the western blotting field and troubleshooting of common problems are highlighted and discussed. This work is a comprehensive primer and guide for new western blotting researchers and those interested in a better understanding of the technique or getting better results.
Topics: Blotting, Western; Immunoblotting; Proteins; Electrophoresis, Polyacrylamide Gel
PubMed: 36971113
DOI: 10.2144/btn-2022-0034 -
Analytical Biochemistry Mar 2020Attaining true quantitative data from WB requires that all the players involved in the procedure are quality controlled including the user. Appropriate protein... (Review)
Review
Attaining true quantitative data from WB requires that all the players involved in the procedure are quality controlled including the user. Appropriate protein extraction method, electrophoresis, and transfer of proteins, immunodetection of blotted protein by antibodies, and the ultimate step of imaging and analyzing the data is nothing short of a symphony. Like with any other technology in life-sciences research, Western blotting can produce erroneous and irreproducible data. We provide a systematic approach to generate quantitative data from Western blot experiments that incorporates critical validation steps to identify and minimize sources of error and variability throughout the Western blot process.
Topics: Blotting, Western; Humans; Limit of Detection; Proteins; Reference Values; Reproducibility of Results
PubMed: 32007473
DOI: 10.1016/j.ab.2020.113608 -
BioTechniques Jun 2022Western blotting (WB), also known as immunoblotting, is a well-known molecular biology method that biologists often use to investigate many features of the protein,... (Review)
Review
Western blotting (WB), also known as immunoblotting, is a well-known molecular biology method that biologists often use to investigate many features of the protein, ranging from basic protein analysis to disease detection. WB is simple, unique, rapid, widely used routine tool with easy interpretation and definite results. It is being used in various fields of science, research and development, diagnostic labs and hospitals. The principle of WB is to accomplish the separation of proteins based on molecular weight and charge. This review addresses in detail the individual steps involved in the WB technique, its troubleshooting, internal loading controls, total protein staining and its diverse applications in scientific research and clinical settings, along with its future perspectives.
Topics: Biomedical Research; Blotting, Western; Proteins; Staining and Labeling
PubMed: 35775367
DOI: 10.2144/btn-2022-0003 -
Biochemistry and Molecular Biology... Jul 2021Western blot (WB) or immunoblot is a workhorse method. It is commonly used by biologists for study of different aspects of protein biomolecules. In addition, it has been... (Review)
Review
Western blot (WB) or immunoblot is a workhorse method. It is commonly used by biologists for study of different aspects of protein biomolecules. In addition, it has been widely used in disease diagnosis. Despite some limitations such as long time, different applications of WB have not been limited. In the present review, we have summarized scientific and clinical applications of WB. In addition, we described some new generation of WB techniques.
Topics: Blotting, Western; Humans; Proteins; Translational Research, Biomedical
PubMed: 33847452
DOI: 10.1002/bmb.21516 -
Angewandte Chemie (International Ed. in... Sep 2019Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin... (Review)
Review
Integrating 2D culture of adherent mammalian cells with single-cell western blotting (in situ scWB) uses microfluidic design to eliminate the requirement for trypsin release of cells to suspension, prior to single-cell isolation and protein analysis. To assay HeLa cells from an attached starting state, we culture adherent cells in fibronectin-functionalized microwells formed in a thin layer of polyacrylamide gel. To integrate the culture, lysis, and assay workflow, we introduce a one-step copolymerization process that creates protein-decorated microwells. After single-cell culture, we lyse each cell in the microwell and perform western blotting on each resultant lysate. We observe cell spreading after overnight microwell-based culture. scWB reports increased phosphorylation of MAP kinases (ERK1/2, p38) under hypertonic conditions. We validate the in situ scWB with slab-gel western blot, while revealing cell-to-cell heterogeneity in stress responses.
Topics: Blotting, Western; Cell Culture Techniques; Humans
PubMed: 31390130
DOI: 10.1002/anie.201906920 -
Frontiers in Immunology 2023Cuproptosis, a newly reported type of programmed cell death, takes part in the regulation of tumor progression, treatment response, and prognosis. But the specific...
BACKGROUND
Cuproptosis, a newly reported type of programmed cell death, takes part in the regulation of tumor progression, treatment response, and prognosis. But the specific effect of cuproptosis-related genes (CRGs) on glioblastoma (GBM) is still unclear.
METHODS
The transcriptome data and corresponding clinical data of GBM samples were downloaded from the TCGA and GEO databases. R software and R packages were used to perform statistical analysis, consensus cluster analysis, survival analysis, Cox regression analysis, Lasso regression analysis, and tumor microenvironment analysis. The mRNA and protein expression levels of model-related genes were detected by RT-qPCR and Western blot assays, respectively.
RESULTS
The expression profile of CRGs in 209 GBM samples from two separate datasets was obtained. Two cuproptosis subtypes, CRGcluster A and CRGcluster B, were identified by consensus cluster analysis. There were apparent differences in prognosis, tumor microenvironment, and immune checkpoint expression levels between the two subtypes, and there were 79 prognostic differentially expressed genes (DEGs). According to the prognostic DEGs, two gene subtypes, geneCluster A and geneCluster B, were identified, and a prognostic risk score model was constructed and validated. This model consists of five prognostic DEGs, including PDIA4, DUSP6, PTPRN, PILRB, and CBLN1. Ultimately, to improve the applicability of the model, a nomogram was established. Patients with GBM in the low-risk cluster have a higher mutation burden and predict a longer OS than in the high-risk group. Moreover, the risk score was related to drug sensitivity and negatively correlated with the CSC index.
CONCLUSION
We successfully constructed a cuproptosis-related prognostic model, which can independently predict the prognosis of GBM patients. These results further complement the understanding of cuproptosis and provide new theoretical support for developing a more effective treatment strategy.
Topics: Humans; Prognosis; Glioblastoma; Nomograms; Apoptosis; Blotting, Western; Tumor Microenvironment
PubMed: 36814929
DOI: 10.3389/fimmu.2023.1082974 -
Molecular Cancer Mar 2023Breast cancer is the most common malignant tumor that threatens women's health. Attention has been paid on the study of long- non-coding RNA (lncRNA) in breast cancer....
BACKGROUND
Breast cancer is the most common malignant tumor that threatens women's health. Attention has been paid on the study of long- non-coding RNA (lncRNA) in breast cancer. However, the specific mechanism remains not clear.
METHODS
In this study, we explored the role of lncRNA BC069792 in breast cancer. In vitro and in vivo functional experiments were carried out in cell culture and mouse models. High-throughput next-generation sequencing technology and real-time fluorescence quantitative PCR technology were used to evaluate differentially expressed genes and mRNA expression, Western blot and immunohistochemical staining were used to detect protein expression. RNA immunoprecipitation assay and dual-luciferase activity assay were used to evaluate the competing endogenous RNAs (ceRNA), and rescue and mutation experiments were used for verification.
RESULTS
We found that lncRNA BC069792 was expressed at a low level in breast cancer tissues, and significantly decreased in breast cancer with high pathological grade, lymph node metastasis and high Ki-67 index groups. Moreover, BC069792 inhibited the proliferation, invasion and metastasis of breast cancer cells in vitro and in vivo. Mechanically, BC069792 acts as a molecular sponge to adsorb hsa-miR-658 and hsa-miR-4739, to up-regulate the protein expression of Potassium Voltage-Gated Channel Q4 (KCNQ4), inhibits the activities of JAK2 and p-AKT, and plays a role in inhibiting breast cancer growth.
CONCLUSIONS
LncRNA BC069792 plays the role of tumor suppressor gene in breast cancer and is a new diagnostic index and therapeutic target in breast cancer.
Topics: Animals; Female; Mice; Blotting, Western; Cell Culture Techniques; Disease Models, Animal; KCNQ Potassium Channels; MicroRNAs; Neoplasms; RNA, Long Noncoding; Humans
PubMed: 36859185
DOI: 10.1186/s12943-023-01747-5 -
Frontiers in Immunology 2023There is a growing public concern about diabetic kidney disease (DKD), which poses a severe threat to human health and life. It is important to discover noninvasive and...
BACKGROUND
There is a growing public concern about diabetic kidney disease (DKD), which poses a severe threat to human health and life. It is important to discover noninvasive and sensitive immune-associated biomarkers that can be used to predict DKD development. ScRNA-seq and transcriptome sequencing were performed here to identify cell types and key genes associated with DKD.
METHODS
Here, this study conducted the analysis through five microarray datasets of DKD (GSE131882, GSE1009, GSE30528, GSE96804, and GSE104948) from gene expression omnibus (GEO). We performed single-cell RNA sequencing analysis (GSE131882) by using CellMarker and CellPhoneDB on public datasets to identify the specific cell types and cell-cell interaction networks related to DKD. DEGs were identified from four datasets (GSE1009, GSE30528, GSE96804, and GSE104948). The regulatory relationship between DKD-related characters and genes was evaluated by using WGCNA analysis. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) datasets were applied to define the enrichment of each term. Subsequently, immune cell infiltration between DKD and the control group was identified by using the "pheatmap" package, and the connection Matrix between the core genes and immune cell or function was illuminated through the "corrplot" package. Furthermore, RcisTarget and GSEA were conducted on public datasets for the analysis of the regulation relationship of key genes and it revealed the correlation between 3 key genes and top the 20 genetic factors involved in DKD. Finally, the expression of key genes between patients with 35 DKD and 35 healthy controls were examined by ELISA, and the relationship between the development of DKD rate and hub gene plasma levels was assessed in a cohort of 35 DKD patients. In addition, we carried out immunohistochemistry and western blot to verify the expression of three key genes in the kidney tissue samples we obtained.
RESULTS
There were 8 cell types between DKD and the control group, and the number of connections between macrophages and other cells was higher than that of the other seven cell groups. We identified 356 different expression genes (DEGs) from the RNA-seq, which are enriched in urogenital system development, kidney development, platelet alpha granule, and glycosaminoglycan binding pathways. And WGCNA was conducted to construct 13 gene modules. The highest correlations module is related to the regulation of cell adhesion, positive regulation of locomotion, PI3K-Akt, gamma response, epithelial-mesenchymal transition, and E2F target signaling pathway. Then we overlapped the DEGs, WGCNA, and scRNA-seq, SLIT3, PDE1A and CFH were screened as the closely related genes to DKD. In addition, the findings of immunological infiltration revealed a remarkable positive link between T cells gamma delta, Macrophages M2, resting mast cells, and the three critical genes SLIT3, PDE1A, and CFH. Neutrophils were considerably negatively connected with the three key genes. Comparatively to healthy controls, DKD patients showed high levels of SLIT3, PDE1A, and CFH. Despite this, higher SLIT3, PDE1A, and CFH were associated with an end point rate based on a median follow-up of 2.6 years. And with the gradual deterioration of DKD, the expression of SLIT3, PDE1A, and CFH gradually increased.
CONCLUSIONS
The 3 immune-associated genes could be used as diagnostic markers and therapeutic targets of DKD. Additionally, we found new pathogenic mechanisms associated with immune cells in DKD, which might lead to therapeutic targets against these cells.
Topics: Humans; Diabetic Nephropathies; Phosphatidylinositol 3-Kinases; Transcriptome; Genes, Regulator; Blotting, Western; Diabetes Mellitus
PubMed: 37063851
DOI: 10.3389/fimmu.2023.1030198 -
Journal of Cellular and Molecular... Jun 2023Molecular profiling has been applied for uterine corpus endometrial carcinoma (UCEC) management for many years. The aim of this study was to explore the role of MCM10 in...
Molecular profiling has been applied for uterine corpus endometrial carcinoma (UCEC) management for many years. The aim of this study was to explore the role of MCM10 in UCEC and construct its overall survival (OS) prediction models. Data from TCGA, GEO, cbioPotal and COSMIC databases and the methods, such as GO, KEGG, GSEA, ssGSEA and PPI, were employed to bioinformatically detect the effects of MCM10 on UCEC. RT-PCR, Western blot and immunohistochemistry were used to validate the effects of MCM10 on UCEC. Based on Cox regression analysis using the data from TCGA and our clinical data, two OS prediction models for UCEC were established. Finally, the effects of MCM10 on UCEC were detected in vitro. Our study revealed that MCM10 was variated and overexpressed in UCEC tissue and involved in DNA replication, cell cycle, DNA repair and immune microenvironment in UCEC. Moreover, silencing MCM10 significantly inhibited the proliferation of UCEC cells in vitro. Importantly, based on MCM10 expression and clinical features, the OS prediction models were constructed with good accuracy. MCM10 could be an effective treatment target and a prognostic biomarker for UCEC patients. The OS prediction models might help establish the strategies of follow-up and treatment for UCEC patients.
Topics: Humans; Female; Prognosis; Treatment Outcome; Blotting, Western; Carcinoma, Endometrioid; Biomarkers; Endometrial Neoplasms; Tumor Microenvironment; Minichromosome Maintenance Proteins
PubMed: 37246638
DOI: 10.1111/jcmm.17772 -
Journal of Veterinary Internal Medicine Mar 2021Fibrinogen heterogeneity has been observed in humans and can influence fibrinogen measurements when using the modified Clauss assay. We hypothesized that fibrinogen...
BACKGROUND
Fibrinogen heterogeneity has been observed in humans and can influence fibrinogen measurements when using the modified Clauss assay. We hypothesized that fibrinogen heterogeneity also exists in horses.
OBJECTIVES
To determine whether fibrinogen heterogeneity exists in horses.
ANIMALS
Five clinically healthy horses from the university equine teaching herd.
METHODS
Presumed fibrinogen was purified from pooled citrated plasma and electrophoresis performed. The purified protein was subjected to Western blotting using sheep antiserum against human fibrinogen, and liquid chromatography-tandem mass spectrometry (LC-MS/MS).
RESULTS
Gel electrophoresis of nonreduced equine purified protein yielded 2 protein bands (approximately 377 and 318 kDa) that corresponded with the molecular weights of human high molecular weight fibrinogen and low molecular weight fibrinogen fractions, respectively. Electrophoretograms of reduced purified protein, Western blots, and LC-MS/MS supported that the purified nonreduced protein bands were fibrinogen.
CONCLUSION
Fibrinogen heterogeneity exists in horses.
Topics: Animals; Blotting, Western; Chromatography, Liquid; Fibrinogen; Horses; Sheep; Tandem Mass Spectrometry
PubMed: 33604912
DOI: 10.1111/jvim.16065