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Signal transduction and exercise
Published in Adam P. Sharples, James P. Morton, Henning Wackerhage, Molecular Exercise Physiology, 2022
Brendan Egan, Adam P. Sharples
Many of the pathways and targets thus far rely heavily on the data from the interrogation of ‘known’ pathways, especially those with well-established canonical roles in cell physiology, in the context of the molecular response to acute exercise. The limitations of methods such as qPCR or Western blotting (Chapter 2) are that only one or a few mRNA or protein targets are measured at a time, and that the choice of these targets is somewhat subjective. An alternative approach is to measure many if not all genetic variations, metabolites, histone modifications, DNA methylations, mRNAs and proteins in an ‘unbiased’ approach. Methods such as metabolomics, epigenomics, gene expression profiling or transcriptomics, and proteomics are examples of unbiased approaches for assessing the changes in metabolites, epigenetic modificatons, mRNA and protein, respectively. Each of these methods generally results in long lists of data that are then analysed and presented using bioinformatics. The above methods can be abbreviated as ‘omic’ approaches. The historical emergence of omics approaches in molecular exercise physiology is described in Chapter 1 and the methods themselves have been covered in more detail in Chapter 2, but here to provide an illustrative example for the advantages of omic approaches, we will briefly cover the relevance of transcriptomics and proteomics in the context of signal transduction pathways.
Diagnostic applications of immunology
Published in Gabriel Virella, Medical Immunology, 2019
Ajay Grover, Virginia Litwin, Gabriel Virella
Western blotting (Figure 15.3) is a technique that identifies specific proteins in a given sample after their separation using polyacrylamide gel electrophoresis. The polyacrylamide gel is placed adjacent to a membrane, which is typically nitrocellulose or PVDF (polyvinylidene fluoride), and the application of an electrical current induces the proteins to migrate from the gel and immobilize on the membrane. The membrane is subsequently stained with an antibody. The confirmatory HIV test employs a Western blot to detect anti-HIV antibody in a human serum sample. Proteins from known HIV-infected cells are separated and blotted on a membrane as discussed previously. Then, the serum to be tested is applied in the primary antibody incubation step; free antibody is washed away, and a secondary antihuman antibody linked to an enzyme signal is added. The stained bands indicate the proteins to which the patient's serum is specific. This technique has the advantage over other screening assays of not only detecting antibodies, but also identifying their target antigens, resulting in increased specificity. However, the procedure is of low throughput and requires specialized personnel in certified laboratories. To circumvent these issues, antigen-specific blots that need only be probed with the patient serum and developed, are now commercially available. Using these commercially available kits, the testing can be conducted in general laboratories.
Prions
Published in Dongyou Liu, Handbook of Foodborne Diseases, 2018
Western blotting is a method in which proteins separated according to molecular weight by sodium dodecyl sulfate polyacrylamide gel electrophoresis are electrically transferred to a membrane for immunological detection. For example, PrP can be detected on the membrane after probing with an anti-PrP antibody (Figure 14.4). If PK treatment is not performed (PK[-]), the same banding pattern is observed for prion infection and noninfection. However, because the N-terminal region of PrP is specifically degraded under PK treatment conditions (PK[+]), the C-terminal region is only detected if PrPres is present. Thus, as the N-terminal region of PrPSc is digested with PK(+), PrPres displays a shift to a lower molecular weight compared to untreated PrP(−) in the Western blot. Moreover, because PrPres patterns detected by Western blotting are different for different prion strains, the technique can be used to classify a particular strain. This observation arises because each prion strain differs in its PK resistance and the modification pattern of the sugar chain.
Overview of gene expression techniques with an emphasis on vitamin D related studies
Published in Current Medical Research and Opinion, 2023
Jeffrey Justin Margret, Sushil K. Jain
Northern blotting, one of the first techniques used to analyze a sample of RNA from a specific cell type or tissue, measures the RNA expression of distinct genes. The expression profile of the gene determined under certain conditions can provide insight into its function7. This relatively laborious method relies on the hybridization between a known nucleic acid probe and the complementary sequence in the mixture of RNAs8. The binding of the probe to the membrane confirms the presence of a complementary RNA sequence in a given sample (Figure 2). These RNA-probe complexes can be detected using different radionuclide labeling. Since this technique uses size-dependent separation, it can only determine the abundance of selected RNA and the size of the transcript9. Northern blotting is a variant of Southern blotting, which is used to analyze DNA, and their protocols are similar. It can also be very effective at detecting novel splice site variants, characterization, and verification of small RNAs, such as miRNAs8. Although its relatively high specificity reduces false positives10, the sensitivity of the Northern blot is diminished if the amount of total RNA or the expression level of the transcript is low9. Recently, the use of Northern blotting in research has been limited due to its low sensitivity, visualization of one or only a few genes at a time, and degradation of the samples by RNases.
Protection of leukemia inhibitory factor against high-glucose-induced human retinal endothelial cell dysfunction
Published in Archives of Physiology and Biochemistry, 2023
Lei Wang, Qiong Wu, Rui Qi Wang, Run Ze Wang, Jianwen Wang
Western blotting was performed using standard methods. Retinal and HREC proteins were precipitated using RIPA buffer and quantified using a protein assay (Bradford Protein Assay; Bio-Rad, USA). Equal amounts of protein were separated by eletrophoresis on 8–12% dodecylsulfate-poluacrylamide gel electrophoresis and electroblotted onto poluvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% skim milk or BSA for 2 hand incubated with antibodies to TNF-α (1:500, Abcam, UK), IL-1β (1:1000, Cell Signalling Technology, USA), IL-6 (1:900, Cell Signalling Technology, USA), VEGF (1:1000, Santa Cruz Biotechnology, USA), HIF-1α (1:1000, Cell Signalling Technology, USA), CaMKII (1:1000, Abcam), p-CaMKII (1:1000, Abcam, UK), CREB (1:1000, Cell Signalling Technology, USA), p-CREB (1:1000, Cell Signalling Technology, USA) overnight at 4 °C. Then the membranes were incubated with secondary antibodies at room temperature for 1 h. GAPDH (1:1000, Zhongshan Goldenbridge Biotechnology, Beijing, China) was used as an internal parameter. Finally, the blots were scanned with a Chemi Doc MP System (Bio-Rad, CA, USA) and the bands were measured by Image J software.
Vitexin protects melanocytes from oxidative stress via activating MAPK-Nrf2/ARE pathway
Published in Immunopharmacology and Immunotoxicology, 2020
Xiao-Sha Li, Xue-Yong Tang, Wei Su, Xin Li
Western blotting was done in a generic manner as described by previous studies. PIG1 cells were lysed followed by protein extraction and quantification by BCA Protein Assay kit (Pierce, Rockford, IL). An appropriate amount of protein was loaded to 10% premade SDS-PAGE gel (BioRad, Hercules, CA) and separated. Protein was then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA) for biochemical assay. Primary and secondary antibodies were used per the vendor’s instructions (Santa Cruz Biotechnology, Santa Cruz, CA). After blocking in a solution of 5% nonfat dry milk diluted in TBS, the membranes were probed and incubated with primary antibodies overnight at 4 °C. After a wash step, the membranes were incubated for 2 h with HRP-conjugated secondary antibodies at room temperature. Bound antibodies were detected using an ECL western blot detection system (Millipore). Quantification of the western blot bands was done by using ImageJ software (National Institute of Health, USA).