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polyvinylidene difluoride membrane


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  • Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane. 1524220

    We have developed a new detection method of deiminated proteins on polyvinylidene difluoride membranes. Citrulline residues in enzymatically deiminated histones were modified by incubating with diacetyl monoxime and antipyrine in a strong acid mixture. The products were injected to rabbits, and the antibodies obtained were affinity-purified using a modified citrulline column. Sample proteins blotted to the membrane were modified in a similar manner and incubated successively with the purified antibody and an alkaline phosphatase-conjugated second antibody. Detection was performed using a chemiluminescent substrate. The method enabled detection of 3-10 fmol of citrulline residues dot blotted as deiminated model proteins. It visualized numerous rat pituitary soluble proteins that had been enzymatically deiminated and Western blotted to the membrane. The data suggest usefulness of the method for detecting deiminated proteins regardless of the backbone protein molecules. Search for deiminated proteins on the Western blots of various rat tissue homogenates detected a single band on that of spinal cord, another band on that of uterus, and multiple bands on those of skin and hair root. The bands in the former two tissue homogenates comigrated with glial fibrillary acidic protein and vimentin, respectively.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    17-347

  • Optimization of a nonradioactive method for consistent and sensitive determination of activated K-ras protein. 16018961

    Accurate measurement of activity of wild-type K-ras protein is important due to its tumor suppressor action in tissues such as lung. A published method by Taylor and co-workers uses plasmid-containing Escherichia coli cells to produce a glutathione-S-transferase/raf-1 ras binding domain (GST-RBD) fusion protein attached to glutathione beads to isolate activated ras protein. We systematically optimized the method before use on lung tissues. Changing the GST-RBD protein induction temperature from the original 37 to 30 degrees C produced a consistently greater yield of fusion protein. To improve stability of the GST-RBD beads so as to perform large-scale experiments, 0.1% NaN(3) was added. NaN(3)-treated beads retained full affinity for at least 24 days. Sensitivity was improved by using a polyvinylidene difluoride membrane rather than nitrocellulose for immunoblotting. We also compared our GST-RBD beads with two commercial assay kits and found that our beads had both superior sensitivity and reduced variability. In summary, our modification of the GST-RBD affinity method to recover activated K-ras greatly increased the yield of fusion protein, prolonged the useful life of GST-RBD beads to at least 24 days, and enhanced detection sensitivity.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    17-218
    Produktbezeichnung:
    Ras Activation Assay Kit

  • Topology of ATP-binding domain of adrenoleukodystrophy gene product in peroxisomes. 8900413

    Adrenoleukodystrophy (X-ALD) is a demyelinating disorder characterized by the accumulation of saturated very-long-chain fatty acids (> C22:0) due to the impaired activity of lignoceroyl-CoA ligase. The gene responsible for the disease was found to code for a 84-kDa peroxisomal integral membrane protein. Its amino acid sequence has high homology with the ATP-binding cassette superfamily of transporters and it is predicted to have six membrane-spanning segments and a putative ATP-binding domain. To define the function of ALDP, we studied the topology of its ATP-binding domain by using antibodies (1D6) against a hydrophobic domain (amino acid residues 279 to 482) and antibodies (Abct) against the C-terminal 15-amino-acid hydrophilic domain (amino acid residues 731 to 745) of ALDP. The observation of punctate fluorescence in permeabilized ALD fibroblasts, using Abct antibodies but not with antibodies against catalase, suggests that the C-terminal segment of ALDP is projected toward the cytoplasm from the peroxisomal membrane. Trypsinization of intact peroxisomes under isotonic conditions abolishes the Abct antibody recognition site, whereas the 1D6 antibodies identify a degradation product of 43-kDa protein that has been protected and retained by the membrane. This again suggests that the C-terminal portion of the ALDP protein is located on the outside (cytoplasmic) face of the peroxisomal membrane. Additional support for this conclusion was obtained by purification of the ALDP C-terminal domain, released from purified rat liver peroxisomes incubated with the cytosolic fraction, using blue-Sepharose affinity chromatography. A 47-kDa peptide retained by the column was recognized by Western blot analysis with Abct antibodies against the C-terminal sequence of ALDP and this polypeptide on polyvinylidene difluoride membrane was able to bind [gamma-32P]ATP in vitro in the presence of Mg2+. These results demonstrate that the C-terminal peptide containing the ATP-binding domains of ALDP is on the cytoplasmic surface of the peroxisomal membrane where this domain may function as an ATPase to support the functional role of ALDP in the peroxisomal membrane.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    MAB2164
    Produktbezeichnung:
    Anti-Adrenoleukodystrophy Protein Antibody, a.a. 279-482, clone 2AL-1D6

  • Retinol-binding protein: a major secretory product of the pig conceptus. 2340335

    Pig conceptuses and endometrial explants recovered from gilts between Days 10 and 15 of pregnancy were cultured in leucine-deficient or methionine-deficient medium supplemented with 3H-leucine or 35S-methionine, respectively, for 30 h. Conceptus and endometrial tissues from Day 15 of pregnancy were fixed in Bouin's fixative for immunocytochemistry and light microscopy. Conceptus culture medium from Day 15 of pregnancy was pooled, dialyzed, and fractionated by anion exchange and gel filtration chromatography. A family of 3-5 low molecular weight (Mr) acidic (Mr = 19,000-22,000; pI = 5.6-6.5) 3H-leu-labeled proteins were isolated and identified by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), electroblotting, and fluorography. The two major proteins (pCSP-1 and pCSP-2) were excised from a polyvinylidene difluoride transfer membrane, and NH2-terminal amino acids were sequenced. One peptide was sequenced through 33 amino acids and the second, which shared 100% homology, was sequenced through 22 amino acids. Analysis of the larger sequence indicated that it shared 93.9% and 90.9% homology with the first 33 amino acids of human and rabbit plasma retinol-binding protein (RBP), respectively. Analyses of culture medium from pig conceptus incubations by 2D-PAGE and immunoprecipitation with rabbit anti-human RBP serum indicated that immunoreactive RBP was produced between Days 10 and 15 of pregnancy and was present in Day 30 allantoic fluid. Western blotting of enriched fractions of Day 15 conceptus RBP followed by immunostaining indicated that five isoforms of radiolabeled RBP were present. Immunoreactive RBP was detected in trophectoderm and yolk sac of conceptuses and endometrial surface and glandular epithelium at Day 15 of pregnancy. Results from this study demonstrate that pig conceptuses secrete RBP prior to onset of conceptus elongation and throughout the peri-implantation period, which suggests that RBP and associated retinoids influence conceptus development.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    20-176
    Produktbezeichnung:
    100X GTPγS, 10mM

  • Processing of the precursor of protamine P2 in mouse. Peptide mapping and N-terminal sequence analysis of intermediates. 1854346

    Protamine P2, the major basic chromosomal protein of mouse spermatozoa, is synthesized as a precursor almost twice as long as the mature protein, its extra length arising from an N-terminal extension of 44 amino acid residues. This precursor is integrated into chromatin of spermatids, and the extension is processed during chromatin condensation in the haploid cells. We have studied processing in the mouse and have identified two intermediates generated by proteolytic cleavage of the precursor. H.p.l.c. separated protamine P2 from four other spermatid proteins, including the precursor and three proteins known to possess physiological characteristics expected of processing intermediates. Peptide mapping indicated that all of these proteins were structurally similar. Two major proteins were further purified by PAGE, transferred to poly(vinylidene difluoride) membranes and submitted to automated N-terminal sequence analysis. Both sequences were found within the deduced sequence of the precursor extension. The N-terminus of the larger intermediate, PP2C, was Gly-12, whereas the N-terminus of the smaller, PP2D, was His-21. Both processing sites involved a peptide bond in which the carbonyl function was contributed by an acidic amino acid.
    Dokumententyp:
    Referenz
    Produkbestellnummer:
    17-191
    Produktbezeichnung:
    MAP Kinase/Erk Assay Kit, non-radioactive
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