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  • Identification of the steroids in neonatal plasma that interfere with 17 alpha-hydroxyprogesterone radioimmunoassays. 1526021

    Neonatal plasma contains interferents that increase the apparent 17 alpha-hydroxyprogesterone (17-OHP) content measured by direct (no-extraction) radioimmunoassay. We fractionated extracts from neonatal plasma pools by liquid chromatography with a Sephadex LH-20 column and measured 17-OHP immunoreactivity by a direct test kit. We found immunoreactivity in the free steroid and glucuronide fraction and also in the monosulfate fraction. We analyzed these two fractions by reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry. We collected fractions and assayed for 17-OHP immunoreactivity. The HPLC fractions containing the interfering steroid monosulfates were analyzed by ion-spray mass spectrometry and, after solvolysis, by gas chromatography-mass spectrometry. Several monosulfates were identified, including those of 17 alpha-hydroxy-pregnenolone, 16 alpha-hydroxypregnenolone, pregnenolone, and several pregnenetriols. 17 alpha-Hydroxypregnenolone sulfate was the most significant interferent. Other commercially available 17-OHP assays showed similar interference when used without an extraction step. Kit manufacturers should select antibodies and protocols to minimize cross-reaction with sulfates, especially 17 alpha-hydroxypregnenolone sulfate.
    문서 타입:
    Reference
    카탈로그 번호:
    20-176
    제품명:
    100X GTPγS, 10mM

  • The nerve growth factor: purification as a 30,000-molecular-weight protein. 4982360

    The nerve growth factor protein was purified over 100-fold from adult mouse salivary glands. The first step was a gel filtration on Sephadex G-100 at pH 7.5 of the aqueous gland extract. After gel filtration, most of the NGF activity was eluted in the 80,000-90,000-molecular-weight region (G-100 pool). The G-100 pool was dialyzed at pH 5.0 and fractionated by CM52 cellulose chromatography at pH 5.0. Recovery from CM52 cellulose columns was quantitative for protein and ranged 80-100 per cent for the nerve growth factor activity; the latter was almost completely carried by a protein which did not show any heterogeneity when examined by several analytical tests. The purified nerve growth factor showed an S(20,w) = 2.43, a D(20,w) = 7.30 and a 30,000 molecular weight. The over-all recovery was about 45 per cent.
    문서 타입:
    Reference
    카탈로그 번호:
    Multiple
    제품명:
    Multiple

  • Cell based radioimmunoassays to quantitate the immunoreactivity of TNT monoclonal antibodies directed against intracellular antigens. 2040706

    Direct and indirect radioimmunoassay (RIA) procedures to determine the amount of binding of a mouse monoclonal antibody (MoAb) reactive with an intracellular antigen present in human cells are described. In these RIAs, mouse IgG2a MoAb, designated as Tumor Necrosis Treatment (TNT-1) antibody, paraformaldehyde/acetone fixed cells, and Sephadex beads were used to standardize the assay conditions. In the direct RIA, 83% of the 125I-labeled TNT-1 MoAb was bound to the target cells within 30 min after the addition of reagents. The amount of binding of the MoAb was directly proportional to the amount of antigen present in the assay. When the direct RIA was carried out using different types of target cells, 125I-labeled TNT-1 MoAb showed greater than 70% binding. In the indirect RIA, the amount of binding of secondary 125I-labeled goat anti-mouse IgG antibody to the target cells was linear. These results suggest that the indirect RIA can be used to estimate the immunoreactivity of the unlabeled TNT-1 MoAb present in crude protein preparations. Based on the results of RIAs the following two conclusions are drawn: 1) the direct RIA can be used to estimate rapidly the amount of immunoreactive TNT-1 MoAb present in 125I-labeled antibody preparations and 2) the indirect RIA which estimates the amount of immunoreactivity of unlabeled TNT-1 MoAb can be used to monitor the purification and study the characteristics of the MoAb present in crude protein preparations. These methods enable the quantitative measurement of MoAbs reactive against intracellular antigens using standard RIA procedures.
    문서 타입:
    Reference
    카탈로그 번호:
    MAB3864
    제품명:
    Anti-DNA/Histone H1 Antibody

  • Identification of multiple in vivo phosphorylation sites in rabbit myelin basic protein. 6185481

    Myelin basic protein of rabbit brain (Mr = 18,200) was initially freed of the bulk of the nonphosphorylated species (mainly component 1) by Cm-cellulose chromatography at high pH. The remainder of the protein was subjected to peptic digestion at pH 6.00, which resulted in specific, essentially complete cleavage at several bonds (Phe-44--Phe-45, Phe-87--Phe-88, Leu-109--Ser-110, and Leu-151--Phe-152) and partial cleavage at the Tyr-14--Leu-15 bond. Gel filtration of the digest through Sephadex G-25 (fine) yielded three fractions, the first containing primarily peptides 1-44 and 45-87, the second peptides 15-44, 88-109, and 110-151, and the third peptides 1-14 and 152-168. Each fraction was chromatographed on Cm-cellulose at pH 8.2, and the resulting subfractions and partially purified peptides were analyzed for phosphoserine and phosphothreonine. Materials containing significant amounts of the phosphoamino acids were subsequently chromatographed on Cm-cellulose at pH 4.65, and the analyses for phosphoserine and phosphothreonine were repeated. The resulting purified peptic phosphopeptides were identified by amino acid analysis and tryptic peptide mapping. Comparison of the maps with those of the unphosphorylated counterparts located the tryptic phosphopeptides. These were recovered and their identities were established by amino acid analysis. In those cases where the phosphopeptide contained 2 Ser residues, the position of the phosphoserine was established by aminopeptidase M digestion. Five phosphorylation sites were found: Ser-7, Ser-56, Thr-96, Ser-113, and Ser-163. Only a small fraction of these sites was phosphorylated in the total basic protein, with values ranging from about 2 (ser-113) to 6% (Thr-96). With the possible exception of Ser-56, these sites are not the ones that have been reported to be phosphorylated in vitro by cyclic AMP-dependent protein kinase.
    문서 타입:
    Reference
    카탈로그 번호:
    Multiple
    제품명:
    Multiple

  • Apolipoprotein A-II content of human plasma high density lipoproteins measured by radioimmunoassay. 198505

    A double antibody radioimmunoassay for human ApoA-II is reported. ApoA-II isolated from human plasma high density lipoprotein (HDL) by column chromatography migrated as a single band on polyacrylamide disc gel electrophoresis, had the appropriate amino acid composition, and provoked the production of monospecific antisera. (125)I-ApoA-II (iodinated by lactoperoxidase, purified by Sephadex G-75 chromatography) migrated with "cold" ApoA-II as a single band on disc gel electrophoresis in SDS. Its specific radioactivity was 5-12 mCi/ micro g. In assays, (0.05 M barbital buffer, 0.01% Triton X-100, pH 8.6) over 90% of (125)I-ApoA-II was bound by excess first antibody and over 95% was displaced by excess "cold" ApoA-II. Low density lipoprotein, very low density lipoprotein, ApoA-I, ApoC-II, and ApoC-III displaced no counts. Intraassay and interassay coefficients of variation for lipoprotein or plasma samples were 7 +/- 4 and 11 +/- 6%, respectively. As little as 1.0 ng of ApoA-II was detectable with a precision of 10%. ApoA-II made up 20-25% of the proteins of HDL (d 1.083-1.19), HDL(2) (d 1.083-1.124), and HDL(3) (d 1.124-1.19) on column chromatography. The ApoA-II contents of these HDL fractions were also 20-25% by radioimmunoassay. Similar results were obtained whether assays were carried out on intact or delipidated HDL samples. Thus, in contrast with ApoA-I (only 10% of which is detectable), all of the ApoA-II contents of intact HDL are detected with accuracy by this assay. Plasma levels of ApoA-II in young normolipemic subjects were approximately 40 mg/dl (n = 29). In these subjects, over 98% of ApoA-II was found in the d 1.063-1.21 density fractions.
    문서 타입:
    Reference
    카탈로그 번호:
    ALP10
    제품명:
    Apolipoprotein A-I, human

  • A rapid method for the preparation of 125I-labelled human growth hormone for receptor studies, using reverse-phase high performance liquid chromatography. 3004459

    Human growth hormone was labelled with 125 Iodine by the stoichiometric modification of the chloramine-T method to a specific activity of 50-80 microCi/microgram, and the iodinated mixture was purified by reverse-phase high performance liquid chromatography using a C18 column (SynChropak RP-P) and a linear gradient. Compared with the usual Sephadex G-100 chromatography, HPLC gave a much better separation, with a higher yield and a considerably reduced analysis time (30 min vs 5 h). The [125I]-labelled preparation had normal binding to IM-9 lymphocyte receptors. The maximum bindability of the HPLC-purified preparation approximated 90%, which is the best value so far reported for human growth hormone. It is concluded that HPLC is a fast, convenient and reproducible method for obtaining an improved [125I]-labelled human growth hormone for receptor studies.
    문서 타입:
    Reference
    카탈로그 번호:
    17-191
    제품명:
    MAP Kinase/Erk Assay Kit, non-radioactive

  • Characterization of proliferating cell nuclear antigen recognized by autoantibodies in lupus sera. 6142919

    A nuclear antigen associated with cell proliferation (proliferating cell nuclear antigen [PCNA]) and blast transformation is recognized by autoantibodies in the sera of some patients with systemic lupus erythematosus. Using this autoantibody as a reagent, PCNA was purified 120-fold by ammonium sulfate fractionation, DEAE chromatography, and Sephadex G200 gel filtration. The antigenicity of PCNA was sensitive to trypsin but resistant to ribonuclease and deoxyribonuclease, suggesting that the antigenic determinant resided in protein and not nucleic acids. PCNA was inactivated at 56 degrees C for 30 min. Isoelectrophoretic focusing showed that the pI was 4.8. Analysis of immunoprecipitates on polyacrylamide gels showed the presence of IgG heavy and light chains and a single polypeptide band of 33,000 mol wt. This polypeptide band was the reactive antigen in immunoblotting (Western transfer) assays.
    문서 타입:
    Reference
    카탈로그 번호:
    Multiple
    제품명:
    Multiple

  • Production of the rat type 1 insulin-like growth factor-binding protein by well differentiated H4EIIC3 hepatoma cells: identification, purification, and N-terminal amino ... 2164920

    We recently identified a 32 K mol wt insulin-like growth factor (IGF)-binding protein (BP) which is markedly increased in the serum of streptozotocin-diabetic rats and recognized by antiserum against the human amniotic fluid IGFBP (hIGFBP-1). In the present study we sought to confirm that this protein represents the rat homolog of IGFBP-1 (rIGFBP-1), and that rIGFBP-1 may, therefore, play an important role in the regulation of IGF bioactivity in experimental diabetes. Since the abundance of related hepatic mRNA is high in diabetic rats, we asked whether well differentiated H4EIIC3 rat hepatoma cells produce rIGFBP-1 and provide sufficient amounts of this protein for purification and further characterization. Specific IGF-binding activity in hepatoma conditioned medium was detected initially by incubation with 125I-labeled recombinant human IGF-II and precipitation with polyethylene glycol. Ligand blotting demonstrated a 32 K BP, identical in size to the major low mol wt IGFBP found in diabetic rat serum. Affinity labeling and immunoprecipitation confirmed that this BP is related to human IGFBP-1 and is distinct from the fetal rat IGFBP, rIGFBP-2. Incorporation of [35S]methionine into 32 K BPs confirmed synthesis by hepatoma cells. For purification of BPs, conditioned medium was collected in roller culture, and BPs were purified by ammonium sulfate precipitation, Sephadex G-75 chromatography, and reverse phase HPLC. Partial amino acid sequencing of purified protein demonstrated 68% identity with the human IGFBP-1 and distinguished this BP from previously characterized rat IGFBPs. Purified protein bound both IGF-I and IGF-II with high affinity. We conclude that the 32 K IGFBP produced by H4EIIC3 hepatoma cells in culture represents the rat form of IGFBP-1 (rIGFBP-1). Regulation of rIGFBP-1 may play an important role in the modulation of IGF bioactivity in experimental animals with metabolic disease. The availability of purified rIGFBP-1 and identification of a cell line that produces this BP will greatly facilitate future studies of IGFBP-1 in the rat model.
    문서 타입:
    Reference
    카탈로그 번호:
    06-580

  • Human kallistatin, a new tissue kallikrein-binding protein: purification and characterization. 2558505

    A new and specific tissue kallikrein-binding protein was identified in mammalian serum and in secreted transformed-cell culture media (Chao et al., Biochem. J. 239: 325-331, 1986). We have designated this kallikrein-binding protein as "kallistatin". Human kallistatin has been purified from serum, using chromatographic steps including DEAE-Sephadex, hydroxylapatite, Cibacron blue-Sepharose, Sephacryl S200, and preparative polyacrylamide gel electrophoresis. The purified kallistatin consists of a single polypeptide chain with an apparent molecular weight of approximately 54 kDa and isoelectric point of approximately 5.0. Kallistatin was eluted as a single peak on reverse-phase HPLC. The purified kallistatin and 125I-labelled human tissue kallikrein form a approximately a 92 kDa SDS- and heat-stable complex. The complex formation is pH dependent and is inhibited by 0.1% (W/V) of deoxycholate or SDS but not by 0.5% (W/V) of Triton X-100, digitonin, Lubrol or CHAPS. A approximately 54 kDa protein was identified in partially purified kallistatin by polyclonal anti-kallistatin antibodies in Western blot analysis and by its binding to 125I-labelled-human tissue kallikrein in ligand blotting. The role of kallistatin in regulating tissue kallikrein activity and metabolism may now be evaluated.
    문서 타입:
    Reference
    카탈로그 번호:
    ABC51

  • Purification and peptide mapping of rat brain choline acetyltransferase. 7430139

    Acetyl-CoA:choline O-acetyltransferase (EC 2.3.1.6) has been purified approximately 17,000-fold from rat brain. The purification protocol included acid and NH4SO4 precipitation, followed by chromatography on CM-Sephadex, phenyl-Sepharose, Sephadex G-150, and blue dextran Sepharose. Two peaks of enzyme activity were detected after blue dextran Sepharose chromatography containing an overall yield of approximately 4% of the starting enzyme activity. The final specific activity of the blue dextran fractions was 70 to 80 mumol of acetylcholine formed min-1 mg of protein-1. Sodium dodecyl sulfate electrophoresis of the blue dextran fractions revealed three closely spaced proteins with a molecular weight of approximately 67,000. Tryptic peptide maps were prepared for each of the gel bands and indicated that they contained nearly identical primary structure.
    문서 타입:
    Reference
    카탈로그 번호:
    MAB305
    제품명:
    Anti-Choline Acetyltransferase Antibody, clone 1E6
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