Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysis

Neuhoff, Volker; Stamm, Reinhard; Eibl, Hansjörg

doi:10.1002/elps.1150060905

Cited by 569 publications

(310 citation statements)

References 30 publications

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“…For immunodetection, a monospecific antibody directed against the recombinant AtStr1 protein was used (Papenbrock and Schmidt, 2000a). The protein spots corresponding to the immunostained proteins were localized on a Coomassie-stained (Neuhoff et al, 1985) two-dimensional gel loaded with 1 mg mitochondrial protein and analyzed by mass spectrometry.…”

Section: Organelle Isolation Sds-page and Western Blottingmentioning

confidence: 99%

Intracellular Localization of Arabidopsis Sulfurtransferases

Bauer

Dietrich²,

Nowak³

et al. 2004

Plant Physiology

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Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one-and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism.All members in the sulfurtransferase (Str)/rhodanese protein family in archaea, eubacteria, and eukaryota are unified by characteristic well-defined sequence domains (Bordo and Bork, 2002). These domains are found as tandem repeats, with the C-terminal domain containing the active site Cys residue, as singledomain proteins or as members of multidomain proteins (Bordo and Bork, 2002). The 18 proteins identified in Arabidopsis which contain at least one Str signature were classified into six groups on the basis of their sequence homology (Bauer and Papenbrock, 2002; http://arabidopsis.org/info/ genefamily/STR_genefamily.html). Group I consists of two Str proteins with two-domain; the five proteins in group VI contain only the C-terminal Str signature and thus possess similarity to the single-domain Str from bacteria.Strs catalyze the transfer of a sulfur atom from suitable sulfur donors to a nucleophilic acceptor. Specific biological roles for most members of this superfamily have not been established (Spallarossa et al., 2001). Proposed roles include cyanide detoxification (Vennesland et al., 1982), involvement in sulfate assimilation (Donadio et al., 1990), and mobilization of sulfur for iron-sulfur cluster biosynthes...

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Section: Organelle Isolation Sds-page and Western Blottingmentioning

confidence: 99%

Intracellular Localization of Arabidopsis Sulfurtransferases

Bauer

Dietrich²,

Nowak³

et al. 2004

Plant Physiology

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“…Gels were then stained for 20 min with 0.1 '/o (w/v) AgNO, and developed with 3 % (w/v) sodium carbonate containing 0.1 '/o (v/v) formaldehyde and development was stopped with glacial acetic acid according to the method of Merril et al (1982). For Coomassie blue staining, gels were fixed in 12% TCA and stained overnight using 0 1 '/o (w/v) purified Coomassie brilliant blue G 250 in 2% (w/v) phosphoric acid, 6% (w/v) ammonium sulphate (Neuhoff et al, 1985). Gels were destained with methanol/water/acetic acid (50 : 40 : 10, by vol.).…”

Section: / Omentioning

confidence: 99%

A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins

1997

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Porphyromonas gingiwalis has been associated with the development of adult periodontitis and cysteine proteinases with trypsin-like specificity have been implicated as major virulence factors. We have extracted the major cellassociated trypsin-like proteolytic activity of P. gingiwalis W50 using mild sonication. Anion-exchange and gel-f iltration FPLC of the sonicate revealed that Arg-and Lys-specific proteinase activity was associated with a 300 kDa complex which could be dissociated into seven bands (48,45,44,39,27,17 and 15 kDa) by SDS-PAGE with the 4 4 kDa band containing two different proteins as shown by N-terminal sequence analysis. On further chromatography of the 300 kDa complex on ArgSepharose the majority of the complex eluted from the affinity column as an undissociated complex. However, a small amount dissociated such that the Lys-and Arg-specific activities could be separated by eluting first with lysine then arginine, respectively. The 45 kDa protein of the complex was purified by further anion-exchange FPLC in the presence of octylpD-glucopyranoside and was shown to be an Arg-specif ic, thiol-activated, calcium-stabilized cysteine proteinase. The 48 kDa protein was also further purified in a similar fashion and shown to be a Lys-specific cysteine proteinase that was not inhibited by EDTA. The two 44 kDa and the 39,27,17 and 15 kDa proteins of the complex exhibit amino acid sequence homology and are proposed to be haemagglutinindadhesins. The 45 kDa Arg-specif ic proteinase and one of the 44 kDa adhesins as well as the 15,17 and 27 kDa adhesins are processed from the single polyprotein encoded by the gene designated prtR, with all proteins preceded by an Arg or Lys residue within the polyprotein. Similarly, the 48 kDa Lys-specific proteinase, the 39 and 15 kDa adhesins as well as the other 44 kDa adhesin of the 300 kDa complex are encoded by a single gene designated prtK, with all proteins preceded by an Arg or Lys residue within the polyprotein. The 39,15 and 44 kDa adhesins of PrtK all exhibit high homology with the 44,15,17 and 27 kDa adhesins encoded by prtR, particularly the 15 kDa proteins which are identical. The cell-associated proteinase-adhesin complex, designated PrtR-PrtK, is therefore composed of the two gene products, the mature PrtR (160 kDa) and mature PrtK (163 kDa) that are further proteolytically processed (most likely autolytically) to release proteinase and adhesin domains that remain non-covalently associated. The fully processed PrtR-PrtK complex comprises the cysteine proteinases PrtR45 and PrtK48 and seven sequence-related adhesin molecules, PrtR44, PrtRl5, PrtRl7, PrtR27 and PrtK39, PrtK15 and PrtK44. We propose that this proteinase-adhesin complex is a major virulence factor for P. gingiwalis On: Sat, 12 May 2018 21:46:32 P. S. BHOGAL, N. SLAKESKI a n d E. C. REYNOLDS involved in the evasion of host defence and in the assimilation of haem and peptides.

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“…SDS/polyacrylamide gel electrophoresis was performed on cast gels from Novex (10-20% acrylamide, TrisRricine system) according to the method of Schagger and von Jagow (1987) and the instructions of the supplier. Protein was stained with Coomassie brillant blue G250 in phosphoric acid (Neuhoff et al, 1985).…”

Section: Purification Of Rldtimentioning

confidence: 99%

Purification, Characterization and Biological Evaluation of Recombinant Leech‐Derived Tryptase Inhibitor (rLDTI) Expressed at High Level in the Yeast Saccharomyces Cerevisiae

Pohlig

Fendrich

Knecht

et al. 1996

European Journal of Biochemistry

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An efficient expressiordpurification procedure has been developed which allows the production of pure, biologically active recombinant leech-derived tryptase inhibitor (rLDTI), originally found in the leech Hirudo medicinalis. The gene for LDTI was generated synthetically from three overlapping oligonucleotides by PCR synthesis. LDTI was expressed in the yeast Saccharomyces cerevisiae under the control of the copper-inducible CUP1 promoter and fused to the invertase signal sequence (SUCZ). The entire expression cassette was inserted into the yeast high-copy vector pDP34. Appropriate host strains transformed with the expression plasmid secreted rLDTI into the medium upon copper addition. Proteinchemical analysis of the secreted rLDTI revealed exclusively inhibitor with the correct N-terminal sequence. Up to 60% of the rLDTI, however, appeared to be modified by glycosylation and the unglycosylated material showed heterogeneity at the C-terminus. Besides full-length rLDTI, truncated rLDTI species lacking either the terminal Asn46 or in addition the penultimate Leu45 were isolated. The C-terminally truncated variants were eliminated using a S. cerevisiae host strain disrupted in the structural genes of carboxypeptidases yscY and ysca, thus identifying these proteases as being responsible for the degradation of rLDTI.Mature rLDTI was purified in high yields from the culture supernatant of the carboxypeptidasedeficient yeast strain by cation-exchange chromatography and reverse-phase HPLC. The recombinant protein is at least 98 % pure, based on HPLC and capillary electrophoresis, and is fully biologically active. Structural identity with the authentic leech protein was confirmed by sequence analysis and molecularmass determination. The purified protein was tested for its ability to inhibit tryptase and trypsin in vitro and to interfere with the tryptase-induced proliferation of human fibroblasts and keratinocytes. Recombinant LDTI appears to be as potent as the authentic leech protein, exhibiting K,-values of ~1 . 5 nM and ~1 . 6 nM against human tryptase and bovine trypsin, respectively. The tryptase-induced proliferation of human fibroblasts and keratinocytes was inhibited with half-maximum values of ~0 . 1 nM and = I nM, respectively. The availability of the recombinant material will allow evaluation of the concept of tryptase inhibition in various disease models and to test the therapeutic potential of LDTI in mast-cell-related disorders.

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Clear background and highly sensitive protein staining with Coomassie Blue dyes in polyacrylamide gels: A systematic analysis

Cited by 569 publications

References 30 publications

Intracellular Localization of Arabidopsis Sulfurtransferases

Intracellular Localization of Arabidopsis Sulfurtransferases

A cell-associated protein complex of Porphyromonas gingivalis W50 composed of Arg- and Lys-specific cysteine proteinases and adhesins

Purification, Characterization and Biological Evaluation of Recombinant Leech‐Derived Tryptase Inhibitor (rLDTI) Expressed at High Level in the Yeast Saccharomyces Cerevisiae

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