Protein transfer to nitrocellulose filters

Vaessen, R.T.M.J.; Kreike, Jan; Groot, G.S.P.

doi:10.1016/0014-5793(81)80134-2

Cited by 209 publications

(51 citation statements)

References 14 publications

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“…Published procedures were used for: manipulation of DNA [12]; transformation of yeast cells [15]; isolation of RNA from yeast [Ih]; isolation of total yeast cell extracts [17]; blotting of RNA to diazobenzyloxymethyl (DBM) paper [18]; protein electrophoresis [19]; protein blotting and immunodecoration [20] and DNA sequence analysis with chain elongation inhibitors [21].…”

Section: Miscellaneousmentioning

confidence: 99%

Nucleotide sequence of the gene encoding the 11‐kDa subunit of the ubiquinol – cytochrome‐c oxidoreductase in Saccharomyces cerevisiae

Maarse

Grivell

1987

European Journal of Biochemistry

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The nucleotide sequence of the gene encoding the 11-kDa subunit VIII of the ubiquinol-cytochrome-c oxidoreductase in Succhuromyces cerevisiue has been determined. The coding sequence has a length of 330 bp and is preceded at a distance of 361 bp by another reading frame, coding for a protein of as yet unknown function. The 1 1-kDa gene is transcribed independently of the URFx gene and transcription of both is sensitive to catabolite repression. Multiple 5' and 3' termini of transcripts of the gene for the 11-kDa subunit were identified by S1 nuclease protection analysis of DNA . RNA hybrids. The 5' termini map 52 f 2 and 60 2 nucleotides upstream of the initiation codon whereas the 3' termini map 336 f 2 and 350 & 2 nucleotides downstream of the stop codon. The subunit VIII reading frame encodes a protein with a molecular mass of 12.4 kDa and a polarity of 37.6%. It is predicted to contain a high content of fl-sheet segments, which may be capable of forming a barrel-like structure in a lipid bilayer. A comparison of the sequence with those of the small subunits of the beef heart complex reveals similarity with the 9.5-kDa subunit VII (core-linked protein) characterized by Borchart et al. (1986) FEBS Lett. 200, 81 -86. The significance of this is discussed.The ubiquinol -cytochrome-c oxidoreductase (complex 111) in Succhuromyces cerevisiue consists of at least 10 different subunits [l] and is part of the respiratory chain. Cytochrome b is the only subunit of the complex that is encoded by mitochondrial DNA, the others are encoded by nuclear DNA. They are synthesized in the cytoplasm, imported into the mitochondrion and assembled together with cytochrome b into a functional complex in the inner membrane. Steady-state levels of the subunits are dependent on the need of the cell for mitochondrial function and previous work has suggested that this coordinate regulation occurs primarily at the level of transcription [2, 31. In order to study this regulation at the molecular level we have cloned a number of genes encoding subunits of complex I11 and determined their nucleotide sequence [2, 4-61. In this article we report the nucleotide sequence of the ll-kDa-protein gene and some of the predicted properties of its product. MATERIALS AND METHODS Strains and growth conditionsRecombinant DNA constructs in M13 vectors were propagated in Escherichia coli JM 103 [7] grown on 2YT medium [1.6% (w/v) bactotrypton (Difco), 1 % (w/v) yeast extract (Gibco), 5% (w/v) NaCl]. Strain EC490 [8] was used for propagation of the shuttle vectors pFLl [9], p285 [2] and pACM1. EC 490 transformants were selected on 2YT medium containing 50 pg/ml ampicillin.Correspondence to L. A.

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Section: Miscellaneousmentioning

confidence: 99%

Nucleotide sequence of the gene encoding the 11‐kDa subunit of the ubiquinol – cytochrome‐c oxidoreductase in Saccharomyces cerevisiae

Maarse

Grivell

1987

European Journal of Biochemistry

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“…The band corresponding to OEC-33 was eluted from the gel, injected into a rabbit and polyclonal antisera prepared [30]. The specificity of the antibody preparation was tested by reaction with proteins from a variety of oxygen-evolving organisms using electroblot radioimmune assay [39].…”

Section: Preparation Of the Antibodiesmentioning

confidence: 99%

“…Maize leaf proteins were subjected to SDS-PAGE [40] and analysed for specific proteins by electroblot radioimmune assay [39]. The nitrocellulose sheets were probed first with antibody and then with "'1-protein A.…”

Section: Electroblot Radioimmune Assaysmentioning

confidence: 99%

Light‐dependent accumulation and localization of photosystem II proteins in maize

Sutton

Sieburth

Bennett

1987

European Journal of Biochemistry

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We have raised antibodies against several major components of photosystem 11. These antisera, which are directed against the apoproteins of two chlorophyll-binding proteins (CPa-1 and CPa-2), the apoprotein of lightharvesting complex I1 and the 33-kDa extrinsic protein of the oxygen-evolving complex, were used to examine the light regulation of photosystem I1 assembly in maize. The principal findings of this study are as follows.1. The 33-kDa protein is present in dark-grown maize and the content increases 5 -10-fold upon illumination. 2. The level of the protein is mediated at least in part by phytochrome and is independent of the accumulation of chlorophyll.3. In contrast, none of the three chlorophyll-binding proteins examined was detectable in leaves of maize grown in darkness or under other light regimes where chlorophyll does not accumulate.4. Even in the absence of photosystem I1 assembly, the 33-kDa protein is properly transported across the thylakoid into the lumen. However, the protein does not attach in the normal way to the inner surface of the membrane under these conditions. Photosystem I1 (PS 11) is one of several multisubunit complexes located in the chloroplast photosynthetic membrane (for review see Murphy [l]). Like the other complexes, which include photosystem I, plastoquinone-plastocyanin oxidoreductase and ATP synthase, PS I1 is composed of both nuclear and chloroplast-encoded proteins. The isolation of intact oxygen-evolving PS I1 particles and of numerous mutants altered in PS I1 composition has begun to provide an understanding of the controls which contribute to the coordination of membrane protein assembly. The PS 11 complex consists of the closely associated LHC I1 lightharvesting polypeptides and a core complex containing the reaction center and several extrinsic proteins involved in oxygen evolution. The major polypeptides of the core are: two Chl-binding proteins, CPa-1 and CPa-2; two putative quinone-binding proteins, D1 and D2; cytochrome b559, and a 24-kDa protein of unknown function [2]. The major extrinsic constituents of the oxygen-evolving complex are a 33-kDa protein and hydroxylamine [9, 12 -141. While the six major proteins of the PS I1 core are chloroplast-encoded [2,15 -201, the three water-oxidizing proteins are coded for by nuclear DNA and must traverse the inner and outer chloroplast membranes as well as the thylakoid membrane prior to assembly. These proteins are synthesized in the cytoplasm as larger precursors [21] and processed in one or several steps [22] to mature size during transport within the chloroplast.The coordination of protein accumulation between the chloroplast and the cytoplasm can occur by a variety of mechanisms. First, the synthesis or accumulation of proteins in the two compartments can be regulated similarly by a common photoreceptor. The two major photoreceptors in plants are phytochrome [23] and protochlorophyllide, whose photoactivation results in chlorophyll synthesis [24]. The synthesis of the nuclear-encoded small subunit and the chlor...

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“…Antibody subclasses were determined by ELISA in Terasaki plates coated with rabbit antisera specific for mouse immunoglobulin subclasses (Miles Laboratories (Vaessen et al, 1981).…”

Section: Neutralizing and Non-neutralizing Monoclonal Antibodies To Tmentioning

confidence: 99%

Neutralizing and Non-neutralizing Monoclonal Antibodies to the E2 Glycoprotein of Semliki Forest Virus Can Protect Mice from Lethal Encephalitis

Boere

Benaissa-Trouw

Harmsen

et al. 1983

Journal of General Virology

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SUMMARYTwo monoclonal antibodies (UM 4.2 and UM 5.1) directed against the glycoprotein E2 of Semliki Forest virus (SFV) are described; both belong to the IgG2a isotype but are of different idiotype. Analysis employing isoelectric focusing resulted in different focusing patterns for both monoclonals (UM 4.2, pI 8; UM 5.1, pI 7.2). They further differed in their ability to neutralize virus. The UM 4.2 antibodies were inactive in neutralization, while the UM 5.1 antibodies exceeded conventional mouse hyperimmune serum in this respect. Both monoclonal antibodies, however, were able to protect mice passively from a lethal infection with SFV. Based on the amount of protein, the UM 5.1 antibodies were 100-fold more effective than the UM 4.2 antibodies in mouse protection tests.Immunization of mice with an avirulent strain of Semliki Forest virus (SFV) results in production of a heterogeneous population of antibodies which are able to interfere with different viral activities like infectivity and haemagglutination (Dalrymple et al., 1976; Helenius et al., 1976). For an analysis of the role played by the individual membrane glycoproteins El, E2 and E 3 of SFV (Garoffet al., 1974), monospecific antibodies are required. We produced a panel of monoclonal antibodies (MA) directed against the glycoproteins E1 and E2. Two MA specific for the E2 glycoprotein but differing in biological properties are studied in this paper.BALB/c mice were immunized with the avirulent SFV strain MRS MP 192/7 and spleen ceils were subsequently fused with P3-NS 1-1Ag4-1 (NS 1) myeloma cells, using the method described by Fazekas de St. Groth & Scheidegger (1980). Hybrids were selected in hypoxanthineaminopterin-thymidine medium (Littlefield, 1964) and antibody-producing clones by plaque titration and enzyme-linked immunosorbent assay (ELISA). Plaque titration and the plaque reduction test (PRT) have been described previously (Kraaijeveld et al., 1979b). ELISA was performed in Terasaki plates (Falcon Plastics) which had been coated with 10 ~tl amounts per well of 0-3 to 0-5 ~tg of purified inactivated SFV in 0.1 M-carbonate-bicarbonate buffer pH 9.6. Samples of hybridoma culture fluid (5 ~tl) were screened for anti-SFV antibody using goat antimouse IgG or IgM antibodies conjugated with alkaline phosphatase (Tago, Burlingame, Ca., U.S.A.). The substrate disodium p-nitrophenyl phosphate (Sigma) was added and the test was scored visually after 10 min. Positive cultures were subcloned by limiting dilution and tested again for anti-SFV activity. Positive clones were injected into pristane-primed female BALB/c mice (0.5 ml pristane intraperitoneally 1 to 2 weeks before injection of cells) at a dose of 1.0 × 106 to 5-0 x 106 cells/mouse. The antibody specificity in the resulting ascitic fluid was identified by immunoblotting (Fig. 1 a). Two clones, UM 4.2 and UM 5.1, showed specificity for the E2 glycoprotein ofSFV. Hyperimmune mouse serum served as a control and reacted with both glycoproteins E1 and E2 as expected.Antibody subclasses were determined by...

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Protein transfer to nitrocellulose filters

Cited by 209 publications

References 14 publications

Nucleotide sequence of the gene encoding the 11‐kDa subunit of the ubiquinol – cytochrome‐c oxidoreductase in Saccharomyces cerevisiae

Nucleotide sequence of the gene encoding the 11‐kDa subunit of the ubiquinol – cytochrome‐c oxidoreductase in Saccharomyces cerevisiae

Light‐dependent accumulation and localization of photosystem II proteins in maize

Neutralizing and Non-neutralizing Monoclonal Antibodies to the E2 Glycoprotein of Semliki Forest Virus Can Protect Mice from Lethal Encephalitis

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