Immunodetection of Proteins on "Western" Blots Using 125I-Labeled Protein A

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.

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Clarithromycin inhibits overproduction of muc5ac core protein in murine model of diffuse panbronchiolitis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00216.2002 ◽

2003 ◽

Vol 285 (4) ◽

pp. L847-L853 ◽

Cited By ~ 46

Author(s):

Yukihiro Kaneko ◽

Katsunori Yanagihara ◽

Masafumi Seki ◽

Misuzu Kuroki ◽

Yoshitsugu Miyazaki ◽

...

Keyword(s):

Core Protein ◽

Periodic Acid ◽

Protein A ◽

Mitogen Activated Protein Kinase ◽

Periodic Acid Schiff ◽

Western Blots ◽

Diffuse Panbronchiolitis ◽

Mucin Production ◽

Long Term Treatment

Long-term treatment of macrolide antibiotics is considered an effective treatment for diffuse panbronchiolitis (DPB). Although hypersecretion is a common feature of this disease, and it is known that macrolides inhibit mucin production, the mechanism of the effect on mucin production is unclear. The aim of our study was to determine the production of muc5ac core protein, a major core protein of mucin in airway secretion, and the effect of clarithromycin treatment on such production in a mouse model mimicking DPB. Alcian blue-periodic acid-Schiff-positive cells were detected in the lungs of Pseudomonas aeruginosa-infected mice. Western blots of these mice showed muc5ac glycoprotein at day 1 and increased progressively from day 4 to day 14 after inoculation of bacteria. Clarithromycin (10 mg · kg-1· day-1for 7 days) significantly reduced the muc5ac expression at both the mRNA and protein levels. To investigate the role of molecules upstream in muc5ac regulation, we examined the role of mitogen-activated protein kinase. Extracellular signal-regulated kinase 1/2 phosphorylation increased in the infected lung and decreased after treatment. Our results suggest that overproduction of muc5ac plays an important role in the pathogenesis of DPB and that clinical improvement following macrolide therapy seems to involve, at least in part, its inhibition of mucin overproduction, through modulation of intracellular signal transduction.

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Quantification of Ag-NOR proteins using Ag-NOR staining on western blots.

Journal of Histochemistry & Cytochemistry ◽

10.1177/42.11.7930534 ◽

1994 ◽

Vol 42 (11) ◽

pp. 1513-1517 ◽

Cited By ~ 15

Author(s):

P Roussel ◽

V Sirri ◽

D Hernandez-Verdun

Keyword(s):

Mammalian Cells ◽

Protein A ◽

Ribosomal Genes ◽

Western Blots ◽

Nucleolar Activity ◽

Cell Extracts ◽

Nucleolar Proteins ◽

Pathological Conditions ◽

Nuclear Extracts ◽

Protein B23

Ribosomal genes are associated with a set of silver-stained nucleolar proteins, the Ag-NOR proteins, whose amount is directly related to the duration of the cell cycle. Quantification of Ag-NOR proteins by image analysis is presently used to evaluate the rate of proliferation of cancer cells and nucleolar activity. Our objective was to establish a procedure to quantify independently each major Ag-NOR protein in cell extracts. Computerized densitometry established that the specific silver staining of Ag-NOR proteins (Ag-NOR staining) performed on Western blots makes it possible to quantify Ag-NOR proteins. Using purified Ag-NOR proteins, nucleolin, and protein B23, we observed that the intensity of Ag-NOR staining is proportional to the amount of protein. A linear relationship exists between the intensity of Ag-NOR staining and the amount of nucleolin, in the range of 0.2-1.6 micrograms. Using total nuclear extracts prepared from mammalian cells, the proportionality was maintained for total Ag-NOR-stained proteins or for a particular protein. We also determined the levels of nuclear proteins suitable for quantitative analysis. Individual Ag-NOR proteins can be quantified by computerized densitometry in nuclear extracts after Ag-NOR staining on Western blots. This procedure can be applied to establish the contribution of each Ag-NOR protein in general staining, estimate the variability of each Ag-NOR protein in normal and pathological conditions, and quantify each Ag-NOR protein contained per cell.

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Exofacial regions of the glucose transporter of human erythrocytes: detection with polyclonal antibodies

Biochemistry and Cell Biology ◽

10.1139/o88-130 ◽

1988 ◽

Vol 66 (10) ◽

pp. 1126-1133 ◽

Cited By ~ 3

Author(s):

Elena Burdett ◽

Amira Klip

Keyword(s):

Metabolic Regulation ◽

Glucose Transporter ◽

Polyclonal Antibodies ◽

Protein A ◽

Glucose Transporters ◽

Human Erythrocytes ◽

Erythrocyte Membranes ◽

Western Blots ◽

Intact Cells ◽

Human Erythrocyte Membranes

The glucose transporter of human erythrocytes is a glycoprotein of 492 amino acids with a Mr of 55 000. From hydrophobicity plots based on the transporter's amino acid sequence, it has been proposed that exofacially, there are only a segment of 34 residues and the glycosylating carbohydrate branch. To detect changes in the number of glucose transporters during metabolic regulation in intact cells, one should obtain antibodies directed to exofacial sites of the transporter. Antibodies to the purified glucose transporter (Band 4.5), intact or deglycosylated with endoglycosidase F, were raised in rabbits. These antibodies, when purified by column chromatography on protein A-Sepharose and by adsorption onto erythrocyte membranes, cross-reacted with the glycosylated glucose transporter on Western blots. The reactivity of the polyclonal antibodies with intact cells was tested by incubating these cells with the antibody, followed by a centrifugation and a subsequent reaction with 125I-labelled goat-antirabbit immunoglobulin G. Intact human erythrocytes reacted positively with the anti-Band 4.5 antibodies but not with nonimmune sera. Reaction with human erythrocytes was about 10 times greater than with pig erythrocytes, which lack glucose transporters. The reaction with intact cells was not due to contamination with broken cells since under the conditions used, broken (freeze–thawed) cells or membranes did not sediment. Reaction with human erythrocyte membranes was more than fivefold higher than with pig erythrocyte membranes. Rat L6 muscle cells reacted with anti-Band 4.5 antibodies; there were about 10 times more binding sites in any one cell in L6 cells than in human erythrocytes, roughly paralleling their relative content of glucose transporters. It is concluded that the antibody may be reacting with exofacial regions of the glucose transporter in intact cells. This suggests that the antibodies may be used to detect relative changes in glucose transporter number on the cell surface.

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Purification and properties of a 23 kDa Ca2+-binding protein from Drosophila melanogaster

Biochemical Journal ◽

10.1042/bj2710661 ◽

1990 ◽

Vol 271 (3) ◽

pp. 661-666 ◽

Cited By ~ 6

Author(s):

L E Kelly

Keyword(s):

Binding Proteins ◽

Binding Protein ◽

Protein A ◽

Mammalian Brain ◽

Binding Studies ◽

Mobility Shift ◽

Western Blots ◽

Heat Stable ◽

Drosophila Protein ◽

Sheep Brain

Recent reports have shown that there exists in mammalian brain a number of heat-stable Ca2(+)-binding proteins that are distinct from calmodulin [McDonald & Walsh (1985) Biochem. J. 232, 559-567]. We have attempted to characterize equivalent Ca2(+)-binding proteins from Drosophila. Affigel-phenothiazine chromatography, which can be used to purify calmodulin and other Ca2(+)-binding proteins, allowed the identification of a possible heat-stable 23 kDa Ca2(+)-binding protein. A purification procedure for this protein has been devised. Purified 23 kDa protein shows characteristics typical of a Ca2(+)-binding protein; there is a mobility shift on SDS/polyacrylamide gels in the presence of EGTA, and Western blotting, followed by the use of the 45Ca2+ overlay technique, confirms that the 23 kDa protein does bind Ca2+. 45Ca2+ binding studies indicate that this protein binds 1 mol of Ca2+/mol of protein, with Kd 1.9 microM. A single band with pI 5.2 is obtained on isoelectric focusing. Analysis of Western blots of Drosophila tissues probed with antibodies to the Ca2(+)-binding protein indicates that it has a widespread distribution, but is absent from muscle tissue. The antibodies also cross-react with a protein of identical molecular mass in extracts of sheep brain. The possible similarity between this Drosophila Ca2(+)-binding protein and mammalian proteins is discussed, and comparison is made between this Drosophila protein and other Ca2(+)-binding proteins purified from vertebrates.

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A novel 49-kilodalton protein from Artemia cross-links microtubules in vitro

Biochemistry and Cell Biology ◽

10.1139/o92-150 ◽

1992 ◽

Vol 70 (10-11) ◽

pp. 1055-1063 ◽

Cited By ~ 3

Author(s):

Jianshe Zhang ◽

Thomas H. MacRae

Keyword(s):

Protein A ◽

Cross Linking ◽

Amino Acid Residues ◽

Microtubule Organization ◽

Western Blots ◽

Microtubule Associated Proteins ◽

Amino Terminal ◽

Apparent Homogeneity ◽

Associated Proteins

A 49 kilodalton (kDa) protein, previously proposed to cross-link microtubules, was purified to apparent homogeneity from cell-free extracts of the brine shrimp Artemia. When incubated with tubulin under assembly conditions, the purified 49-kDa protein cross-linked the resulting microtubules. Preformed microtubules were also cross-linked when incubated with the 49-kDa protein. Upon centrifugation through sucrose cushions the 49-kDa protein cosedimented with microtubules, suggesting a stable association between the cross-linking protein and tubulin. Such microtubules were interconnected by particles which were circular, bilobed, or elongated in shape. Disruption of microtubule cross-linking and dissociation of the 49-kDa protein from microtubules occurred in the presence of ATP and 5′-adenylylimidodiphosphate (AMP–PNP), a nonhydrolyzable analogue of ATP. The 49-kDa protein was moderately resistant to heat, it did not stimulate tubulin assembly, and it did not react with antibodies to neural microtubule-associated proteins (MAPs) and kinesin. These observations indicate that the 49-kDa protein is different from many known MAPs, a conclusion strengthened by the inability of antibodies raised to the 49-kDa protein to recognize these proteins. The amino terminal 15 amino acid residues of the 49-kDa protein were determined by Edman digestion and an antibody raised to this peptide reacted with the 49-kDa protein on Western blots. Microtubule cross-linking was unaffected by the synthetic amino-terminal peptide, even when it was present at a fivefold molar excess over the 49-kDa protein. A search of three protein databanks revealed that the amino terminus of the 49-kDa protein is unique among published sequences. The findings verify our earlier proposal that Artemia contains a 49-kDa microtubule cross-linking protein and demonstrate that it has a novel set of characteristics. The 49-kDa protein has the potential to play an important role in microtubule organization and function.Key words: microtubule cross-linking, microtubule-associated proteins, Artemia.

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A Novel Brighter Bioluminescent Fusion Protein Based on ZZ Domain and Amydetes vivianii Firefly Luciferase for Immunoassays

Frontiers in Bioengineering and Biotechnology ◽

10.3389/fbioe.2021.755045 ◽

2021 ◽

Vol 9 ◽

Author(s):

Vadim R. Viviani ◽

Jaqueline Rodrigues Silva ◽

Paulo Lee Ho

Keyword(s):

Fusion Protein ◽

Western Blotting ◽

Protein A ◽

Firefly Luciferase ◽

Western Blots ◽

Promising Alternative ◽

Ccd Imaging ◽

Atp Assay ◽

Luminescent Signal ◽

Secondary Antibodies

Immunoassays are widely used for detection of antibodies against specific antigens in diagnosis, as well as in electrophoretic techniques such as Western Blotting. They usually rely on colorimetric, fluorescent or chemiluminescent methods for detection. Whereas the chemiluminescence methods are more sensitive and widely used, they usually suffer of fast luminescence decay. Here we constructed a novel bioluminescent fusion protein based on the N-terminal ZZ portion of protein A and the brighter green-blue emitting Amydetes vivianii firefly luciferase. In the presence of D-luciferin/ATP assay solution, the new fusion protein, displays higher bioluminescence activity, is very thermostable and produces a sustained emission (t1/2 > 30 min). In dot blots, we could successfully detect rabbit IgG against firefly luciferases, Limpet Haemocyanin, and SARS-CoV-2 Nucleoprotein (1–250 ng), as well as the antigen bound antibodies using either CCD imaging, and even photography using smartphones. Using CCD imaging, we could detect up to 100 pg of SARS-CoV-2 Nucleoprotein. Using this system, we could also successfully detect firefly luciferase and SARS-CoV-2 nucleoprotein in Western Blots (5–250 ng). Comparatively, the new fusion protein displays slightly higher and more sustained luminescent signal when compared to commercial HRP-labeled secondary antibodies, constituting a novel promising alternative for Western Blotting and immunoassays.

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Trafficking of immature ΔF508-CFTR to the plasma membrane and its detection by biotinylation

Biochemical Journal ◽

10.1042/bj20081869 ◽

2009 ◽

Vol 419 (1) ◽

pp. 211-221 ◽

Cited By ~ 17

Author(s):

Yishan Luo ◽

Ken McDonald ◽

John W. Hanrahan

Keyword(s):

Cystic Fibrosis ◽

Plasma Membrane ◽

Epithelial Cells ◽

Molecular Mass ◽

Protein A ◽

Airway Epithelial Cells ◽

Mutant Form ◽

Western Blots ◽

Δf508 Cftr ◽

R Domain

Recent studies suggest that immature, core-glycosylated ΔF508-CFTR [the predominant mutant form of the CFTR (cystic fibrosis transmembrane conductance regulator)] can reach the plasma membrane under some conditions. In the present study we investigated this possibility since it has implications for understanding how therapeutics rescue the trafficking of mutant CFTR and perhaps other misfolded proteins. Core-glycosylated CFTR was labelled and pulled down on streptavidin beads after exposure to sulfo-NHS-SS-biotin [biotin attached to a reactive NHS (N-hydroxysuccinimide) ester with a disulfide spacer; molecular mass=606.7 Da]; however, intracellular proteins were also detected in the precipitates. When the R domain of CFTR was expressed in the cytosol of BHK (baby-hamster kidney) cells as a soluble polypeptide it was also labelled after surface biotinylation and pulled down on streptavidin beads. Intracellular biotinylation was reduced when cells were treated with sulfo-NHS-LC-biotin (biotin attached to a reactive NHS ester with an aminocaproic acid spacer) or sulfo-NHS-PEO12-biotin [biotin attached to a reactive NHS ester with a poly(ethylene glycol) spacer], but the reduction could be explained by the lower reactivity of these reagents. The R domain was detected on Western blots after loading <0.25% of the pulldown sample (∼0.01% of total lysate protein), a fraction that could be ascribed to cells that were permeable to ethidium homodimer-1 (molecular mass=856.8 Da) and propidium iodide (molecular mass=668.6 Da). When BHK cells were incubated at 29 °C to rescue ΔF508-CFTR trafficking, and then biotinylated and sorted to remove permeable cells, labelling of core-glycosylated ΔF508-CFTR was no longer detected although a weak signal was still observed using CFBE (cystic fibrosis bronchial epithelial) cells. These results suggest that there is weak surface expression of immature ΔF508-CFTR on airway epithelial cells and demonstrate the need to remove permeable cells when studying CFTR glycoforms by surface biotinylation.

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Cytochemical localization of ATP diphosphohydrolase from Leishmania (Viannia) braziliensis promastigotes and identification of an antigenic and catalytically active isoform

Parasitology ◽

10.1017/s0031182009991661 ◽

2009 ◽

Vol 137 (5) ◽

pp. 773-783 ◽

Cited By ~ 9

Author(s):

F. A. REZENDE-SOARES ◽

C. CARVALHO-CAMPOS ◽

M. J. MARQUES ◽

G. N. PORCINO ◽

N. L. L. GIAROLA ◽

...

Keyword(s):

Protein A ◽

Lower Amount ◽

Serum Samples ◽

Igg Antibody ◽

Cytochemical Localization ◽

Western Blots ◽

Atp Diphosphohydrolase ◽

Catalytically Active ◽

Human Immune Response ◽

Elisa Technique

SUMMARYAn ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83% ATPase and 87% of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1:50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80% seropositivity) and its potato apyrase counterpart (50% seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response.

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Telomere Function and the G-Quadruplex Formation are Regulated by hnRNP U

Cells ◽

10.3390/cells8050390 ◽

2019 ◽

Vol 8 (5) ◽

pp. 390 ◽

Cited By ~ 2

Author(s):

Hiroto Izumi ◽

Keiko Funa

Keyword(s):

Mammalian Cells ◽

Binding Protein ◽

Protein A ◽

Western Blots ◽

Telomere Repeat ◽

G Quadruplex ◽

Hnrnp U ◽

Telomere Biology

We examine the role of the heterogenous ribonucleoprotein U (hnRNP U) as a G-quadruplex binding protein in human cell lines. Hypothesizing that hnRNP U is associated with telomeres, we investigate what other telomere-related functions it may have. Telomeric G-quadruplexes have been fully characterized in vitro, but until now no clear evidence of their function or in vivo interactions with proteins has been revealed in mammalian cells. Techniques used were immunoprecipitation, DNA pull-down, binding assay, and Western blots. We identified hnRNP U as a G-quadruplex binding protein. Immunoprecipitations disclosed that endogenous hnRNP U associates with telomeres, and DNA pull-downs showed that the hnRNP U C-terminus specifically binds telomeric G-quadruplexes. We have compared the effect of telomere repeat containing RNA (TERRA) on binding between hnRNP U and telomeric (Tel) or single- stranded Tel (ssTel) oligonucleotides and found that ssTel binds stronger to TERRA than to Tel. We also show that hnRNP U prevents replication protein A (RPA) accumulation at telomeres, and the recognition of telomeric ends by hnRNP suggests that a G-quadruplex promoting protein regulates its accessibility. Thus, hnRNP U-mediated formation has important functions for telomere biology.

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