scholarly journals The Design of a Quantitative Western Blot Experiment

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Sean C. Taylor ◽  
Anton Posch
Keyword(s):  
Western Blot ◽  
Western Blotting ◽  
Dynamic Range ◽  
Complex Mixture ◽  
Experimental Methodology ◽  
Western Blots ◽  
Western Blot Experiment ◽  
Fundamental Shift ◽  
Western Blot Data ◽  
Densitometric Data

Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

scholarly journals Emerging techniques of western blotting for purification and analysis of protein

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Krishna Kumar Singh ◽  
Anshika Gupta ◽  
Charu Bharti ◽  
Himanchal Sharma
Keyword(s):  
Capillary Electrophoresis ◽  
Western Blot ◽  
Western Blotting ◽  
Blot Analysis ◽  
Clinical Relevance ◽  
Western Blot Analysis ◽  
Molecular Techniques ◽  
Clinical Implications ◽  
Western Blots ◽  
Main Text

Abstract Background Western blotting is frequently employed in molecular techniques like Proteomics and Biology. Because it is a sequential framework, differences and inaccuracies could even take place at any stage, decreasing this particular method's reproducibility and reliability. Main text New approaches, like automated microfluid western blotting, DigiWest, single cell resolution, microchip electrophoresis, and capillary electrophoresis, were all implemented to reduce the future conflicts linked with the western blot analysis approach. Discovery of new in devices and higher susceptibility for western blots gives innovative opportunities to expand Western blot’s clinical relevance. The advancements in various region of west blotting included in this analysis of transfer of protein and validation of antibody are described. Conclusion This paper describes another very developed strategy available as well as demonstrated the correlation among Western blotting techniques of the next generation and their clinical implications. In this review, the different techniques of western blotting and their improvement in different stages have been discussed.

scholarly journals Western Blotting (WB) Technique Guideline for Separation and Isolation of Protein

2021 ◽  
Vol 5 (2) ◽  
pp. 359-372
Author(s):  
Rachmat Hidayat ◽  
Patricia Wulandari
Keyword(s):  
Western Blot ◽  
Western Blotting ◽  
Complex Mixture ◽  
Solid Support ◽  
Target Proteins ◽  
Secondary Antibodies

A B S T R A C TWestern blotting is an important technique used in cell and molecularbiology. Using the western blot, researchers can identify specific proteinsfrom the complex mixture of proteins extracted from cells. This techniqueuses three elements to accomplish this task: (1) separation by size, (2)transfer to a solid support, and (3) marking target proteins using appropriateprimary and secondary antibodies to visualize. This paper will attempt toexplain the techniques and theory behind western blot, and offer severalways to solve the problem

scholarly journals Misleading Westerns: Common Quantification Mistakes in Western Blot Densitometry and Proposed Corrective Measures

2019 ◽  
Vol 2019 ◽  
pp. 1-15 ◽  
Author(s):  
Trent A. J. Butler ◽  
Jonathan W. Paul ◽  
Eng-Cheng Chan ◽  
Roger Smith ◽  
Jorge M. Tolosa
Keyword(s):  
Quality Control ◽  
Western Blot ◽  
Mathematical Models ◽  
Total Protein ◽  
Protein S ◽  
Protein Abundance ◽  
Western Blots ◽  
Sds Page ◽  
Control Protein ◽  
Western Blot Data

Densitometry data generated for Western blots are commonly used to compare protein abundance between samples. In the last decade, it has become apparent that assumptions underpinning these comparisons are often violated in studies reporting Western blot data in the literature. These violations can lead to erroneous interpretations of data and may contribute to poor reproducibility of research. We assessed the reliability of Western blot data obtained to study human myometrial tissue proteins. We ran dilution series of protein lysates to explore the linearity of densitometry data. Proteins analysed included αSMA, HSP27, ERK1/2, and GAPDH. While ideal densitometry data are directly proportional to protein abundance, our data confirm that densitometry data often deviate from this ideal, in which case they can fit nonproportional linear or hyperbolic mathematical models and can reach saturation. Nonlinear densitometry data were observed when Western blots were detected using infrared fluorescence or chemiluminescence, and under different SDS-PAGE conditions. We confirm that ghosting artefacts associated with overabundance of proteins of interest in Western blots can skew findings. We also confirm that when data to be normalised are not directly proportional to protein abundance, it is a mistake to use the normalisation technique of dividing densitometry data from the protein-of-interest with densitometry data from loading control protein(s), as this can cause the normalised data to be unusable for making comparisons. Using spiked proteins in a way that allowed us to control the total protein amount per lane, while only changing the amount of spiked proteins, we confirm that nonlinearity and saturation of densitometry data, and errors introduced from normalisation processes, can occur in routine assays that compare equal amounts of lysate. These findings apply to all Western blot studies, and we highlight quality control checks that should be performed to make Western blot data more quantitative.

Use of monoclonal and polyclonal antibodies to detect prostatic acid phosphatase by immunoblotting.

1986 ◽  
Vol 32 (10) ◽  
pp. 1832-1835 ◽  
Author(s):  
P C Patel ◽  
L Aubin ◽  
J Côte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Western Blot ◽  
Polyclonal Antibodies ◽  
Prostatic Acid Phosphatase ◽  
The Other ◽  
Dot Blot ◽  
Western Blots ◽  
Dot Blot Assay ◽  
Polyclonal Antisera

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.

scholarly journals A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

2021 ◽  
Vol 22 (4) ◽  
pp. 2141
Author(s):  
Srinu Tumpara ◽  
Elena Korenbaum ◽  
Mark Kühnel ◽  
Danny Jonigk ◽  
Beata Olejnicka ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Western Blotting ◽  
Complex Mixture ◽  
Immunoglobulin M ◽  
Confocal Laser ◽  
Enzyme Linked Immunosorbent Assay ◽  
Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

Western blot protocol for detecting ATP10B in mouse/rat brain v1

2021 ◽  
Author(s):  
María Sanchiz Calvo ◽  
eduard.bentea not provided ◽  
Veerle Baekelandt
Keyword(s):  
Western Blot ◽  
Rat Brain ◽  
Brain Tissue ◽  
Western Blotting ◽  
Mouse Brain

Protocol for detection of ATP10B in rat and mouse brain tissue by Western blotting

scholarly journals Evaluating Strategies to Normalise Biological Replicates of Western Blot Data

PLoS ONE ◽  
2014 ◽  
Vol 9 (1) ◽  
pp. e87293 ◽  
Author(s):  
Andrea Degasperi ◽  
Marc R. Birtwistle ◽  
Natalia Volinsky ◽  
Jens Rauch ◽  
Walter Kolch ◽  
...  
Keyword(s):  
Western Blot ◽  
Western Blot Data

scholarly journals Simultaneous identification of Trypanosoma cruzi surface and internal antigens reactive to different immunoglobulin classes (radio-immunoblotting)

1990 ◽  
Vol 32 (5) ◽  
pp. 379-383 ◽  
Author(s):  
Anna Maria Simonsen Stolf ◽  
Eufrosina Setsu Umezawa ◽  
Bianca Zingales
Keyword(s):  
Trypanosoma Cruzi ◽  
Western Blot ◽  
Chagas Disease ◽  
Western Blotting ◽  
Specific Antibodies ◽  
Internal Parasite ◽  
Sds Page ◽  
Blotting Technique ◽  
Simultaneous Identification ◽  
Acute Chagas Disease

A radioactive Western-blotting technique was developed by which the reactivity of Immunoglobulins (Igs) from different classes to both membrane radiolabelled and internal parasite antigens is simultaneously identified. The method includes radioiodination of parasites, polypeptide fractionation by SDS-PAGE, Western-blot transfer and autoradiography of the immunoblots developed with anti-Igs conjugates labelled with enzymes. The analysis is then performed by the comparison of common bands on the autoradiograms and the respective substrate stained nitrocellulose blots. This technique was used to analyse T. cruzi trypomastigote surface labelled antigens reactive to IgM, IgA and IgG specific antibodies. A different pattern of reactivity with acute Chagas' disease patients sera was thus obtained.

A Comparison of an Enzyme Linked Immunosorbent Assay (ELISA) and Western Blotting for Detection of Peanut Mottle Virus and Peanut Stripe Virus1

1986 ◽  
Vol 13 (2) ◽  
pp. 64-67 ◽  
Author(s):  
J. L. Sherwood ◽  
H. A. Melouk
Keyword(s):  
Western Blotting ◽  
Immunosorbent Assay ◽  
Protein A ◽  
Mottle Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Coat Proteins ◽  
Western Blots ◽  
Peanut Stripe Virus ◽  
Dry Milk ◽  
The Difference

Abstract Western blotting was used to detect infections of peanut cv. Tamnut 74 with peanut mottle virus (PMV) and/or peanut stripe virus (PStV). Leaf samples were ground in electrophoresis sample buffer and heated for 5 min at 95 C prior to electrophoresis in 12% polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose sheets at 100V for 45 min. Western blots were performed by first blocking unbound sites on the nitrocellulose with 5% non-fat dry milk in Tris-buffered saline (TBS), pH 7.4 for 30 min, followed by incubation in a 1/200 dilution of PMV and/or PStV antiserum in TBS (the latter antiserum provided by J. W. Demski, U. of GA) for 45 min. This was followed by incubation in protein-A-peroxidase (2 μg/mL in TBS) for 45 min, followed by 4-chloro-1-napthol plus hydrogen peroxide in TBS. As little as 25 ng of either purified PMV or PStV was detected. This was similar to the limits of detection fo the double sandwich enzyme linked immunosorbent assay (ELISA). Because of the difference in migration of the coat proteins of PMV and PStV, both viruses may be detected in plants infected with PMV and PStV. This assay can be performed in approximately 6 h when mini-gels are used for the initial electrophoretic seperation and does not require the antiserum to be fractionated or bound to an enzyme as is the case with ELISA.

scholarly journals Aldosterone-dependent and -independent regulation of Na+ and K+ excretion and ENaC in mouse kidneys

2020 ◽  
Vol 319 (2) ◽  
pp. F323-F334
Author(s):  
Lei Yang ◽  
Gustavo Frindt ◽  
Yuanyuan Xu ◽  
Shinichi Uchida ◽  
Lawrence G. Palmer
Keyword(s):  
Glomerular Filtration Rate ◽  
Western Blot ◽  
Filtration Rate ◽  
Ex Vivo ◽  
Western Blots ◽  
High K ◽  
Collecting Ducts ◽  
Low K ◽  
Clearance Methods

We investigated the regulation of Na+ and K+ excretion and the epithelial Na+ channel (ENaC) in mice lacking the gene for aldosterone synthase (AS) using clearance methods to assess excretion and electrophysiology and Western blot analysis to test for ENaC activity and processing. After 1 day of dietary Na+ restriction, AS−/− mice lost more Na+ in the urine than AS+/+ mice did. After 1 wk on this diet, both genotypes strongly reduced urinary Na+ excretion, but creatinine clearance decreased only in AS−/− mice. Only AS+/+ animals exhibited increased ENaC function, assessed as amiloride-sensitive whole cell currents in collecting ducts or cleavage of αENaC and γENaC in Western blots. To assess the role of aldosterone in the excretion of a K+ load, animals were fasted overnight and refed with high-K+ or low-K+ diets for 5 h. Both AS+/+ and AS−/− mice excreted a large amount of K+ during this period. In both phenotypes the excretion was benzamil sensitive, indicating increased K+ secretion coupled to ENaC-dependent Na+ reabsorption. However, the increase in plasma K+ under these conditions was much larger in AS−/− animals than in AS+/+ animals. In both groups, cleavage of αENaC and γENaC increased. However, Na+ current measured ex vivo in connecting tubules was enhanced only in AS+/+ mice. We conclude that in the absence of aldosterone, mice can conserve Na+ without ENaC activation but at the expense of diminished glomerular filtration rate. Excretion of a K+ load can be accomplished through aldosterone-independent upregulation of ENaC, but aldosterone is required to excrete the excess K+ without hyperkalemia.

Sign in / Sign up

Export Citation Format

Share Document