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 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 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 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.

Study on the interactions between protein disulfide isomerase and target proteins, using immobilization on solid support

FEBS Letters ◽  
1998 ◽  
Vol 426 (1) ◽  
pp. 107-110 ◽  
Author(s):  
Vladimir I Muronetz ◽  
Nian Xian Zhang ◽  
Igor G Bulatnikov ◽  
Chih-Chen Wang
Keyword(s):  
Protein Disulfide Isomerase ◽  
Solid Support ◽  
Disulfide Isomerase ◽  
Target Proteins ◽  
Protein Disulfide

scholarly journals Erythropoietin production by the kidney and the liver in response to severe hypoxia evaluated by Western blotting with deglycosylation

2020 ◽  
Author(s):  
Yukiko Yasuoka ◽  
Takashi Fukuyama ◽  
Yuichiro Izumi ◽  
Yushi Nakayama ◽  
Hideki Inoue ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Western Blot ◽  
Western Blotting ◽  
Fold Increase ◽  
Interstitial Cells ◽  
Severe Hypoxia ◽  
Liquid Chromatography Mass Spectrometry ◽  
Western Blot Method ◽  
Erythropoietin Production

AbstractThe detection of erythropoietin (Epo) protein by Western blotting has required pre-purification of the sample. We developed a new Western blot method to detect plasma and urinary Epo using deglycosylation. Epo in urine and tissue and erythropoiesis-stimulating agents (ESAs) in urine were directly detected by our Western blotting. Plasma Epo and ESAs were detected by our Western blotting after deglycosylation. The broad bands of Epo and ESAs were shifted to 22 kDa by deglycosylation except PEG-bound epoetin β pegol. The 22 kDa band from anemic patient urine was confirmed by Liquid Chromatography/Mass Spectrometry (LC/MS) to contain human Epo.Sever hypoxia (7% O2, 4 hr) caused a 400-fold increase in deglycosylated Epo expression in rat kidneys, which is consistent with the increases in both Epo gene expression and plasma Epo concentration. Immunohistochemistry showed Epo expression in nephrons but not in interstitial cells under control conditions, and hypoxia increased Epo expression in interstitial cells but not in tubules.These data show that intrinsic Epo and all ESAs can be detected by Western blot either directly in urine or after deglycosylation in blood, and that the kidney is the main and sole site of Epo production in control and severe hypoxia. Our method will completely change Epo doping and detection.

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 Combined Use of Indirect ELISA and Western Blotting with Recombinant Hepatocellular Carcinoma-Associated Antigen 59 Is a Potential Immunodiagnostic Tool for the Detection of Prepatent Haemonchus contortus Infection in Goat

Animals ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 548 ◽  
Author(s):  
Muhammad Ali-ul-Husnain Naqvi ◽  
Sana Zahra Naqvi ◽  
Muhammad Ali Memon ◽  
Kalibixiati Aimulajiang ◽  
Muhammad Haseeb ◽  
...  
Keyword(s):  
Western Blot ◽  
Western Blotting ◽  
Haemonchus Contortus ◽  
Indirect Elisa ◽  
Fasciola Hepatica ◽  
False Negative ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Combined Use

Haemonchus contortus is recognized as one of the important health problems in small ruminants, leading to reduced production and economic loss for farmers worldwide. Prepatent diagnosis of H. contortus infection is crucial to improve control strategies as this helminth may remove up to one-fifth of total erythrocytes and may cause anemia, edema, diarrhea, and ultimately death in young animals. In this study, one of the excretory and secretory products, rHc-HCA59, was purified and used as antigen to detect specific antibodies in H. contortus infected goats during prepatent stage of infection using indirect enzyme linked immunosorbent assay (ELISA) as screening test. All goats (n = 38) were housed indoor, experimentally infected with 8000 infective larvae (L3) of H. contortus, and serum samples were collected prior to infection and at 14th day of infection. Immunoblotting was performed to confirm the results of indirect ELISA, evaluate the cross reactivity against rHc-HCA59 in sera of most common co-infecting parasites and rectify the false negative samples. Furthermore, three different batches of rHc-HCA59 were produced to evaluate the repeatability of ELISA. No eggs were detected in feces of all goats collected at 7th and 14th day of infection but, H. contortus eggs were detected at 21 days post infection in the feces. Indirect ELISA performed in this study showed 87% sensitivity and 100% specificity. The western blot analysis confirmed immunoreactivity in serum samples which scored positive in indirect ELISA and recognized the samples as negative which had OD450 lower than negative cut-off value in indirect ELISA. Furthermore, all false negative sera (n = 5) that had OD450 value between positive and negative cut-off value in rHc-HCA59 based ELISA were clearly positive in western blot. Moreover, no cross-reactivity was detected in ELISA and western blotting against rHc-HCA59 in positive sera of Toxoplasma gondii, Fasciola hepatica, and Trichinella spiralis. The results of this study concluded that combined use of indirect ELISA and western blotting with rHc-HCA59 is a potential immunodiagnostic tool for the detection of H. contortus infection during prepatent period in goats.

scholarly journals Interleukin-37 contributes to the pathogenesis of gout by affecting PDZ domain-containing 1 protein through the nuclear factor-kappa B pathway

2020 ◽  
Vol 48 (9) ◽  
pp. 030006052094871
Author(s):  
Wei Wan ◽  
Yeqing Shi ◽  
Lianmei Ji ◽  
Xiaofang Li ◽  
Xia Xu ◽  
...  
Keyword(s):  
Western Blot ◽  
Western Blotting ◽  
Protein Level ◽  
Nuclear Factor Kappa B ◽  
Mrna Level ◽  
Pdz Domain ◽  
Nuclear Factor Κb ◽  
Pyrrolidine Dithiocarbamate ◽  
Nuclear Factor ◽  
Translational Level

Objective Our objective was to explore the molecular pathogenesis of the onset of gout and the mechanism underlying the effect of interleukin (IL)-37 on PDZ domain-containing 1 (PDZK1) protein through the nuclear factor-κB signaling pathway. Methods Real-time PCR and western blotting were used to detect expression of PDZK1 mRNA and protein, respectively, in the HK-2 cell line. The inhibitors pyrrolidine dithiocarbamate (PDTC) and wortmannin were added to HK-2 cells stimulated by IL-37, and changes in PDZK1 protein were detected by western blotting. Results Based on our previous research, we used 10 µmol/L PDTC. We detected no significant change in PDZK1 at the mRNA level among the IL-37, PDTC+IL-37, and wortmannin+IL-37 groups. With increasing IL-37 concentration, the protein level of PDZK1 increased. After adding wortmannin, the protein level of PDZK1 increased with increasing concentration of IL-37, albeit not significantly, and the level of PDZK1 remained lower than that with IL-37 alone. After adding PDTC, the protein level of PDZK1 showed a trend to decrease with increasing concentrations of IL-37 up to 40 ng/mL. The immunofluorescence results supported the western blot results. Conclusions IL-37 can affect protein expression of PDZK1, but not at the translational level, in the pathogenesis of gout.

A longitudinal study of porcine serological responses to experimental infections with T-1 and T-3 SpanishTrichinellaisolates

1992 ◽  
Vol 66 (3) ◽  
pp. 231-237 ◽  
Author(s):  
F. Bolas ◽  
E. Albarran ◽  
T. Garate ◽  
R. M. E. Parkhouse ◽  
A. R. Martinez
Keyword(s):  
Body Weight ◽  
Longitudinal Study ◽  
Antibody Response ◽  
Western Blot ◽  
Western Blotting ◽  
Blot Analysis ◽  
Antigen Recognition ◽  
Gene Pools ◽  
Experimental Infections ◽  
Antibody Levels

ABSTRACTComparison of antibody response and antigen recognition was made by ELISA and western-blot analysis in pig experimental infections by T-l and T-3 SpanishTrichinellaisolates. Two groups of Iberian pigs were experimentally infected with 150 larvae/kg body weight of GM-1 and C-76 SpanishTrichinellaisolates as representatives of T-1 and T-3 gene pools respectively. Antibody levels and antigen recognition were measured on days –14, 0, 6, 16, 20, 27, 34, 49, 63 and 82 after infection by ELISA and western-blotting assays. Antibody response against C-76 infection was significantly delayed and lower than against GM-1. The twoTrichinellaisolates were indistinguishable, however, by western blotting analysis, although recognition of larval antigens was quantitatively higher than adult ones. Interestingly, the principle larval antigenic components recognized by pigs were those recognized by the monoclonal antisera NIM-M1. Finally, there were no serological patterns indicative of the stage of infection (“antibody windows”) discriminating, for example between early versus late infections.

Sign in / Sign up

Export Citation Format

Share Document