Article
Biochemical Research Methods
Masataka Nakagawa, Yui Tomioka, Chiaki Sakuma, Yasunori Kurosawa, Takashi Shibata, Tsutomu Arakawa, Teruo Akuta
Summary: A new protocol for conducting two-dimensional (2D) electrophoresis was developed by combining agarose native gel electrophoresis with sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) or flat SDS agarose gel electrophoresis. The technique utilizes His/MES buffer during the first-dimensional agarose native gel electrophoresis to visualize basic and acidic proteins in their native states or complex structures. The gel strip from the first-dimensional agarose gel electrophoresis is soaked in SDS and placed on top of vertical SDS-PAGE gels or flat SDS-MetaPhor high-resolution agarose gels for the second-dimensional separation.
Article
Polymer Science
Dishiwen Liu, Haoliang Wu, Shengyu Cui, Qingyan Zhao
Summary: Western blotting, a widely used technique in the biomedical field, has been criticized for its time consumption, multiple steps, and low method results. In this study, we modified various steps, including gel preparation, electrophoresis, electrotransfer, blocking, and gel cutting, to improve efficiency and reduce costs. Our modifications shortened the overall experimental time to 80 minutes, increased productivity, and maintained compatibility with existing equipment.
Review
Biochemical Research Methods
Rasheed Sule, Gabriela Rivera, Aldrin Gomes
Summary: Western blotting is a powerful technique for detecting or semiquantifying individual proteins from complex mixtures of cell or tissue extracts. This article provides a comprehensive overview of the history, theory, protocol, and applications of western blotting. It also discusses lesser known but significant problems in the field and troubleshooting techniques. This work serves as a primer and guide for new western blotting researchers and those seeking a better understanding of the technique or improved results.
Article
Biochemistry & Molecular Biology
Chiaki Sakuma, Tomomi Sato, Takashi Shibata, Masataka Nakagawa, Yasunori Kurosawa, C. J. Okumura, Toshiaki Maruyama, Tsutomu Arakawa, Teruo Akuta
Summary: This report demonstrates a simple protocol for blotting agarose native gel to a PVDF membrane and provides three examples of protein analysis. The research shows the potential application of agarose native gel electrophoresis in specific protein studies.
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
(2021)
Article
Biochemical Research Methods
Amber N. Rogers, Maya K. Mastronardo, Tsion G. Mekonnen, Ana Maria Soto
Summary: RNA research is an active field that often requires large amounts of purified RNA. One common purification method involves using electroelution to recover RNA from gel pieces. In this study, two electroeluters with different chamber designs were compared and both were found to effectively recover comparable amounts of purified RNA.
ANALYTICAL BIOCHEMISTRY
(2021)
Article
Biochemical Research Methods
Christoph J. Burckhardt, John D. Minna, Gaudenz Danuser
Summary: Co-immunoprecipitation is widely used in studying protein-protein interactions, but requires considerable optimization, and results are often presented non-quantitatively. This protocol offers an optimized workflow using semi-quantitative immunoblot densitometry for increased reliability and reproducibility.
Article
Biochemical Research Methods
Makoto Hagiwara
Summary: Researchers added acid dyes to polyacrylamide gel electrophoresis to facilitate sample loading and visualization. This simple method can be adopted worldwide for protein analysis.
ANALYTICAL BIOCHEMISTRY
(2022)
Article
Agriculture, Dairy & Animal Science
Larissa Di Marzo, Joice Pranata, David M. Barbano
Summary: The study compared the estimation of milk casein percentage using SDS-PAGE and Kjeldahl methods, finding that Kjeldahl underestimated the proteolytic damage to casein in milk.
JOURNAL OF DAIRY SCIENCE
(2021)
Article
Biochemical Research Methods
Phyllis A. Rees, R. Joel Lowy
Summary: This study examines the performance of Western blot analysis and finds that normalization using the sum of target protein values combined with analytical replication most effectively reduces variability, resulting in low coefficient of variation (CV) and maximal to minimal value (Max/Min) ratios as low as 5-10% and 1.1.
ANALYTICAL BIOCHEMISTRY
(2023)
Article
Engineering, Environmental
Dongqi Wang, Yueyun Li, Helen A. Cope, Xiaoxiao Li, Peisheng He, Cong Liu, Guangyu Li, Sheikh M. Rahman, Nicholas B. Tooker, Charles B. Bott, Annalisa Onnis-Hayden, Jyoti Singh, Alistair Elfick, Ricardo Marques, Henning J. Jessen, Adrian Oehmen, April Z. Gu
Summary: This study demonstrated the use of single-cell Raman spectroscopy (SCRS) for characterizing intracellular polyPs in PAOs within complex environmental samples like EBPR systems. SCRS revealed distinct distribution patterns of polyP length in different PAO types and configurations of EBPR systems, as well as distinctive polyP features among PAO phenotypic sub-groups, indicating its potential for microdiversity-level analysis within EBPR communities.
Article
Multidisciplinary Sciences
Renato Lima Senra, Camilo Jose Ramirez-Lopez, Marcos Jorge Magalhaes-Junior, Joao Gabriel da Silva Neves, Edvaldo Barros, Bruna Waddington, Simone Eliza Facioni Guimaraes, Jose Domingos Guimaraes, Maria Cristina Baracat-Pereira
Summary: This study found that kallikrein proteoforms are involved in the seasonality of horse reproduction. During the breeding season, increased abundance and different types of kallikreins were observed, along with lower sperm motility, increased semen volume, and higher concentrations of glucose and cholesterol.
SCIENTIFIC REPORTS
(2022)
Article
Biochemical Research Methods
Chiaki Sakuma, Masataka Nakagawa, Yui Tomioka, Toshiaki Maruyama, Kevin Entzminger, Jonathan K. Fleming, Takashi Shibata, Yasunori Kurosawa, C. J. Okumura, Tsutomu Arakawa, Teruo Akuta
Summary: This study introduces a Western blotting method for native proteins separated on agarose gels, using contact (diffusion) blotting to transfer proteins onto PVDF membranes. This method avoids denaturation and has successfully detected various native proteins and macromolecular complexes.
Article
Plant Sciences
Mate Sagi-Kazar, Eva Sarvari, Barnabas Cseh, Levente Illes, Zoltan May, Csaba Hegedus, Attila Barocsi, Sandor Lenk, Katalin Solymosi, Adam Solti
Summary: The uptake of iron by chloroplasts does not solely rely on photosynthetic functions, as demonstrated by the ability of etioplasts to acquire iron even in the absence of photosynthetic activity. This likely occurs through the internal reducing capacity of etioplasts supporting the reduction-based iron uptake.
FRONTIERS IN PLANT SCIENCE
(2023)
Article
Food Science & Technology
Yan Zhang, Shengsheng Li, Lizhu Zhao
Summary: The aim of this study was to investigate the influence of different processing methods on the quality, protein oxidation, and structural properties of yak meat. Various parameters such as cooking loss, shear force, color, texture, thiobarbituric acid reactive substance (TBARS), total carbonyl content (TCC), total sulfhydryl content (TSC), and structural properties were measured. The results showed that frying at 80 degrees C resulted in the lowest cooking loss and shear force, indicating better textural characteristics. Drying led to the highest protein oxidation, while frying showed the lowest protein oxidation and the best quality.
JOURNAL OF TEXTURE STUDIES
(2023)
Article
Medicine, Research & Experimental
Museer A. Lone, Mari J. Aaltonen, Aliza Zidell, Helio F. Pedro, Jonas A. Morales Saute, Shalett Mathew, Payam Mohassel, Carsten G. Bonnemann, Eric A. Shoubridge, Thorsten Hornemann
Summary: This study discovered different variants in the SPTLC1 gene that lead to distinct clinical presentations in Amyotrophic lateral sclerosis (ALS). The mutations impair the binding of ORMDL to the holoenzyme complex, resulting in changes in sphingolipid synthesis and lipid characteristics. Additionally, the mutations cause peripheral hereditary sensory and autonomic neuropathy type 1 (HSAN1).
JOURNAL OF CLINICAL INVESTIGATION
(2022)
