[29] Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis

Tsang, Victor C. W.; Peralta, José Mauro; Simons, Adrian

doi:10.1016/0076-6879(83)92032-3

Cited by 627 publications

(238 citation statements)

References 7 publications

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“…The fractionated proteins were electroblotted to 0·45 nm pore size nitrocellulose transfer membranes (SMI) at 300 mA for 45 min [24]. Membranes were stained with Ponceau S to demonstrate successful electrophoresis and transfer of proteins onto the membranes.…”

Section: Electrophoresis and Immunoblottingmentioning

confidence: 99%

Detection and identification of a soy protein component that cross-reacts with caseins from cow's milk

Rozenfeld

Docena

Añón

et al. 2002

Clinical and Experimental Immunology

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Food allergy is becoming a medical, economical and social problem. Soybean, together with milk, peanuts and eggs, are the major allergenic foods. This pathology affects the infant population, when the gut barrier is immature and the immune system is still refining its ability to tolerate food proteins. In our country, cow's milk allergy (CMA) constitutes the main food allergy in infancy, but soy allergy has become exacerbated because of the increased utilization of soy-based formulas as cow's milk (CM) substitutes and the inclusion of soy-proteins in many processed foods. Once a food allergy is diagnosed, the only proven therapy is the strict elimination of the offending allergens from the diet. Available substitutes for CM include milks from different mammalian animals, soy-based formulas, hydrolysed cow's milk proteins (CMP) and amino acid-based formulas. Furthermore, there is evidence that soy proteins may trigger allergic reactions in CMA patients, as assessed by a double-blind placebo controlled food challenge (DBPCFC) [1].Milk contains more than 50 proteins and the major ones are implicated in a number of immunologically mediated reactions [2]. Caseins and b-lactoglobulin (b-Lg) have been described as the main antigenic and allergenic components for human beings [3,4], although immunoglobulins, a-lactalbumin (a-La), and bovine serum albumin (BSA) were also found to be reactive with different isotypes of human antibodies [5,6]. Several immunoreactive epitopes have been identified in caseins [7,. Most of the epitopes present in the casein molecules are sequential since the high number of proline and hydrophobic residues (45%) in these proteins determine an undefined secondary and tertiary structure [10]. Besides, caseins tend to aggregate as a result of hydrophobic interactions to give quaternary structures [11], and this may create conformational epitopes buried in the hydrophobic interior of the micelle [7]. These epitopes are only exposed and accessible to the immune system after denaturation of the complex by digestion.A number of soy proteins that bind antibodies, especially IgE, have been identified [12,13]. The main fractions of storage globulins from seed proteins are the complexes 7S or b-conglycinin, and 11S or glycinin. The 7S complex is generally a trimer with MW 150-200 kD, whereas 11S is a hexamer with MW 300-400 kD [14]. In the 7S fraction, or b-conglycinin, several IgE-binding proteins have been identified and some of them have been purified and characterized: Gly m Bd 28K, Gly m Bd 30K, Gly m Bd 60K [15,16]. Sequence homology has been reported between these allergens and the thiol proteinase family SUMMARYSoy-based formulas are the most employed cow's milk substitutes in the treatment of cow's milk allergy in our country. Since adverse reactions have been reported in allergic patients as a consequence of exposure to soy proteins, we have investigated the possible cross-reactivity between components from soybean and cow's milk. A cow's milk specific polyclonal antiserum and casein specific m...

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Section: Electrophoresis and Immunoblottingmentioning

confidence: 99%

Detection and identification of a soy protein component that cross-reacts with caseins from cow's milk

Rozenfeld

Docena

Añón

et al. 2002

Clinical and Experimental Immunology

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“…SDS-PAGE analysis showed that 35 transformants clearly produced lPTG-inducible random proteins with the expected molecular weight of about 14-16 kDa. The Western blot analysis [8] of the cells of the randomly chosen 25 transformants from the 35 showed that the lPTG-inducible protein bands on SDS-PAGE contain the epitope tag. Some of the results are shown in…”

Section: Rm~ts and Discussionmentioning

confidence: 99%

“…The cells before induction served as the negative control, The detected additional distinct band with an expected molecular weight (Mr 14000-16000) were judged to be the random protein produced by cells after IPTG induction. The presence of random proteins were also identified by Western blot analysis [8] using a monoclonal antibody directed against the epitope tag (Novagen, Inc., Madison, Wl).…”

Section: Expression Identification and In Rive Solubility Of Randommentioning

confidence: 99%

Solubility of artificial proteins with random sequences

et al. 1996

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A library of artificial random proteins of 141 amino acid residues of which 95 are random and which includes the 20 kinds of amino adds was prepared. Out of the 25 identified random proteins, 5 were soluble in the cell lysate, indicating that about 20% of the random proteins expressed in £schericMa ¢oli are expected to be soluble. The soluble random proteins RP3-42 and RP3-45 and insoluble RP3-70 were purified. The solubility of the purified form is the same as that in the cell lysate.

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“…SDS-PAGE and western blotting were done as reported previously (Schagger and von Jagow, 1987;Tsang et al, 1983). HM-1 was detected using anti-HM-1 rabbit polyclonal antibody as the first antibody and alkaline phosphatase-conjugated antirabbit IgG as the secondary antibody.…”

Section: Sds-page and Western Blottingmentioning

confidence: 99%

Genome‐wide screen of Saccharomyces cerevisiae for killer toxin HM‐1 resistance

Miyamoto

Furuichi

Komiyama

2010

Yeast

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We screened a set of Saccharomyces cerevisiae deletion mutants for resistance to killer toxin HM-1, which kills susceptible yeasts through inhibiting 1,3-beta-glucan synthase. By using HM-1 plate assay, we found that eight gene-deletion mutants had higher HM-1-resistance compared with the wild-type. Among these eight genes, five -ALG3, CAX4, MNS1, OST6 and YBL083C -were associated with N -glycan formation and maturation. The ALG3 gene has been shown before to be highly resistant to HM-1. The YBL083C gene may be a dubious open reading frame that overlaps partially the ALG3 gene. The deletion mutant of the MNS1 gene that encodes 1,2-alpha-mannosidase showed with a 13-fold higher HM-1 resistance compared with the wild-type. By HM-1 binding assay, the yeast plasma membrane fraction of alg3 and mns1 cells had less binding ability compared with wild-type cells. These results indicate that the presence of the terminal 1,3-alpha-linked mannose residue of the B-chain of the N -glycan structure is essential for interaction with HM-1. A deletion mutant of aquaglyceroporin Fps1p also showed increased HM-1 resistance. A deletion mutant of osmoregulatory mitogen-activated protein kinase Hog1p was more sensitive to HM-1, suggesting that high-osmolarity glycerol pathways plays an important role in the compensatory response to HM-1 action.

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[29] Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis

Cited by 627 publications

References 7 publications

Detection and identification of a soy protein component that cross-reacts with caseins from cow's milk

Detection and identification of a soy protein component that cross-reacts with caseins from cow's milk

Solubility of artificial proteins with random sequences

Genome‐wide screen of Saccharomyces cerevisiae for killer toxin HM‐1 resistance

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