Production and purification of synthetic peptide antibodies

Nivison, Helen T.; Hanson, Maureen R.

doi:10.1007/bf02668993

Cited by 21 publications

(13 citation statements)

References 23 publications

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“…The cysteine was added to the N terminus of the peptide to provide a sulfhyryl group that was used for conjugation of the peptide to keyhole limpet hemocyanin by m-maleimidobenzoyl N-hydroxysuccinimide ester (20). The conjugated protein was used as an immunogen to raise antibodies in rabbits.…”

Section: Methodsmentioning

confidence: 99%

Molecular Cloning and Functional Analysis of Polyphosphoinositide-dependent Phospholipase D, PLDβ, from Arabidopsis

Pappan

Khatiwada

Dyer

et al. 1997

Journal of Biological Chemistry

108

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A novel plant phospholipase D (PLD; EC 3.1.4.4) activity, which is dependent on phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and nanomolar concentrations of calcium, has been identified in Arabidopsis. This report describes the cloning, expression, and characterization of an Arabidopsis cDNA that encodes this PLD. We have designated names of PLD␤ for this PIP 2 -dependent PLD and PLD␣ for the previously characterized PIP 2 -independent PLD that requires millimolar Ca 2؉ for optimal activity. The PLD␤ cDNA contains an open reading frame of 2904 nucleotides coding for a 968-amino acid protein of 108,575 daltons. Expression of this PLD␤ cDNA clone in Escherichia coli results in the accumulation of a functional PLD having PLD␤, but not PLD␣, activity. The activity of the expressed PLD␤ is dependent on PIP 2 and submicromolar amounts of Ca 2؉ , inhibited by neomycin, and stimulated by a soluble factor from plant extracts. Sequence analysis reveals that PLD␤ is evolutionarily divergent from PLD␣ and that its N terminus contains a regulatory Ca 2؉ -dependent phospholipid-binding (C2) domain that is found in a number of signal transducing and membrane trafficking proteins.Phospholipase D (PLD; EC 3.1.4.4) 1 -catalyzed hydrolysis of glycerophospholipids produces phosphatidic acid (PA) and a hydrophilic constituent. This activity was first identified in plants and since has been found in animals and microorganisms. PLD in plants was originally proposed to be important in phospholipid catabolism, initiating a lipolytic cascade in membrane deterioration during senescence and stress injuries (1, 2). Recent studies in plants, animals, and yeast indicate that PLD hydrolysis plays a pivotal role in transmembrane signaling and cellular regulation (3-9). Activation of PLD has been proposed to mediate many cellular processes including cell proliferation, membrane trafficking, meiosis, and responses to external and internal stimuli. It has been suggested that multiple forms of PLD are involved in these diverse cellular processes since several studies have shown the presence of PLD variants that are expressed differently (9 -12). In castor bean (9) and rice (12), one PLD variant is constitutive whereas the appearance of other variants is associated with specific conditions such as rapid growth, wounding, and senescence. A distinct property shared by these variants is their in vitro requirement of millimolar Ca 2ϩ concentrations for optimal activity. Further analyses of the castor bean PLD variants have led to the suggestion that the catalytic activity of these variants results from the same gene product (9 -11).A recent study has provided important evidence for the presence of two plant PLDs that are derived from different gene products and regulated distinctly (13). One PLD requires polyphosphoinositides and submicromolar concentrations of Ca 2ϩ for activity and the other is PIP 2 -independent and is most active in the presence of millimolar amounts of Ca 2ϩ . The latter is the prevalent form of PLD that has been purified and cha...

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Section: Methodsmentioning

confidence: 99%

Molecular Cloning and Functional Analysis of Polyphosphoinositide-dependent Phospholipase D, PLDβ, from Arabidopsis

Pappan

Khatiwada

Dyer

et al. 1997

Journal of Biological Chemistry

108

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“…The synthetic peptides were conjugated to the maleimide-activated carrier protein keyhole limpet hemocyanin, as described previously (Nivison and Hanson, 1987).…”

Section: Preparation Of Anti-csccd and Anti-cszcd Antibodies And Protmentioning

confidence: 99%

Oxidative Remodeling of Chromoplast Carotenoids

et al. 2002

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The accumulation of three major carotenoid derivatives-crocetin glycosides, picrocrocin, and safranal-is in large part responsible for the color, bitter taste, and aroma of saffron, which is obtained from the dried styles of Crocus. We have identified and functionally characterized the Crocus zeaxanthin 7,8(7 ,8 )-cleavage dioxygenase gene ( CsZCD ), which codes for a chromoplast enzyme that initiates the biogenesis of these derivatives. The Crocus carotenoid 9,10(9 ,10 )-cleavage dioxygenase gene ( CsCCD ) also has been cloned, and the comparison of substrate specificities between these two enzymes has shown that the CsCCD enzyme acts on a broader range of precursors. CsZCD expression is restricted to the style branch tissues and is enhanced under dehydration stress, whereas CsCCD is expressed constitutively in flower and leaf tissues irrespective of dehydration stress. Electron microscopy revealed that the accumulation of saffron metabolites is accompanied by the differentiation of amyloplasts and chromoplasts and by interactions between chromoplasts and the vacuole. Our data suggest that a stepwise sequence exists that involves the oxidative cleavage of zeaxanthin in chromoplasts followed by the sequestration of modified water-soluble derivatives into the central vacuole.

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“…Blots were then incubated in probe solutions containing the primary antibody diluted in blocking buffer overnight (for anti-COXII antibodies) or for 36 hr to 48 hr (for anti-URF-S antibody). When anti-peptide antibodies were used, keyhole limpet hemocyanin (KLH, from Calbiochem) was included in the probe solution (Nivison and Hanson, 1987). Blots were washed for 15 min four times in Tris-saline, blocked for 15 min, and then incubated for 3 hr with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG antibody, both from HyClone).…”

Section: Lmmunological Detection Of Protein On Blotsmentioning

confidence: 99%

“…The 13 (COXII peptide) or 12 (URF-S peptide) amino acid sequences were preceded by a spacer of 3 glycines, a tyrosine for iodination, and a cysteine for coupling. Peptides were coupled to KLH using the cross-linking reagent mmaleimidobenzoyl N-hydroxysuccinimide ester (Pierce) or to BSA using succinimidyl 4-(N-maleimidomethyl)cyclohexane-1 -carboxylate (Pierce) as described (Nivison and Hanson, 1987). KLHpeptide conjugates were injected into rabbits, and antisera were collected.…”

Section: Peptides and Antibodiesmentioning

confidence: 99%

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Identification of a mitochondrial protein associated with cytoplasmic male sterility in petunia.

1989

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The petunia fused gene ( pcf ), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxll, and an unidentified reading frame termed urfS. To determine whether the pcf gene is expressed at the protein level, we produced antibodies to synthetic peptides specified by the coxll and urfs portions of the pcf gene. Anti-COXII peptide antibodies recognized petunia COXll but no other mitochondrial proteins. Anti-URF-S peptide antibodies recognized a 20-kilodalton protein present in both cytoplasmic male sterile and fertile lines and a protein with an apparent molecular mass of 25 kilodaltons present only in cytoplasmic male sterile lines.The 25-kilodalton protein was found to be synthesized by isolated mitochondria and to fractionate into both the soluble and membrane portions of disrupted mitochondria, whereas the 20-kilodalton protein was found only in the membrane fraction. The abundance of the 25-kilodalton protein was much lower in fertile plants carrying the cytoplasmic male sterile cytoplasm and a single dominant nuclear fertility restorer gene, Rf. Thus, the pcf gene is correlated with cytoplasmic male sterility not only by its co-segregation with the phenotype in somatic hybrids, but also by the modification of its expression at the protein level through the action of a nuclear gene that confers fertility.

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Production and purification of synthetic peptide antibodies

Cited by 21 publications

References 23 publications

Molecular Cloning and Functional Analysis of Polyphosphoinositide-dependent Phospholipase D, PLDβ, from Arabidopsis

Molecular Cloning and Functional Analysis of Polyphosphoinositide-dependent Phospholipase D, PLDβ, from Arabidopsis

Oxidative Remodeling of Chromoplast Carotenoids

Identification of a mitochondrial protein associated with cytoplasmic male sterility in petunia.

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