Cyclic Nucleotide Metabolism and Action During Senescence
Richard C. Adelman, George S. Roth in Endocrine and Neuroendocrine Mechanisms of Aging, 2017
The most commonly used method of detecting specific protein phosphorylation involves separation of 32P-labeled proteins by Polyacrylamide gel electrophoresis and quantitation by slicing and counting or autoradiography and densitometry. Labeling may be done by incubation of tissue with phosphate-free media containing carrier-free 32Pi, followed by treatment with the appropriate stimulus, or by incubation of homogenates or subcellular fractions with [33Ρ]-γ-ΑΤΡ of high specific activity in the presence and absence of a cyclic nucleotide or Ca2+. In either case, incubations are terminated as quickly as possible, usually by heating with added sodium dodecyl sulfate (SDS).108 Some distinction between particulate and cytosolic proteins can be made by homogenizing labeled tissue with a buffered solution containing sucrose, NaF (50 mM), NaHP04 (10 mM), EDTA (10 mM), and protease inhibitors to preserve states of phosphorylation. A major problem incurred with intact cells exists in the 32P-RNA that is generated and appears as a high background in the gels, thus interfering with measurement of low level phosphorylation of minor proteins. Extraction with the above solution of inhibitors plus NP-40, a detergent which does not readily solubilize nuclear but does solubilize plasma membranes, and removal of nuclei by centrifugation may partially alleviate this problem. Alternatively, treatment of samples with an RNAse that remains active in 0.3% SDS and mercaptoethanol may also help to reduce this back-ground.109
Biochemistry Of The Murine Ia Antigens
Soldano Ferrone, Chella S. David in Ia Antigens, 2019
Both limited proteolytic digestion and detergent solubilization have been employed in obtaining murine Ia antigens for study. The majority of laboratories studying the biochemistry of murine Ia antigens have employed detergents to solubilize the Ia Antigens. Although both ionic and nonionic detergents will dissolve the lipid bilayer and thus solubilize the Ia Antigens, most workers in the field have chosen to use nonionic detergents. The most frequent choice is Nonidet® P-40 (NP-40) with Triton® X-100 as a second choice. Solubilization of plasma membranes with nonionic detergents produces intact Ia molecules including the membrane-binding portions of the a and (β chains and the associated Ii chain.
Distribution and Biological Functions of Pyruvate Carboxylase in Nature
D. B. Keech, J. C. Wallace in Pyruvate Carboxylase, 2018
Latency of the mitochondrial pyruvate carboxylase activity to its substrates is readily overcome by disruption of the inner mitochondrial membrane by freeze-drying,57 by freezing and thawing,366 by sonication, or with detergents. Both non-ionic detergents (viz., 0.5% NP-40;16 0.06 to 0.5% Triton X-100;518,700,746 Lubrol WX [0.5 to 1 mg/mg protein]173) and deoxycholate (0.1 to 1.0%)212,246,808 have been used successfully, but there is species variation in the optimum concentration to be used.805
S3Ab, a novel antibody targeting B lymphocytes, is a potential therapeutic agent for B-lineage malignancies
Published in Journal of Drug Targeting, 2019
Sisi Li, Hongqiang Shen, Qiang Shu
We isolated the antigen recognised by S3 antibody through immunoprecipitation (IP). First, the S3 antibody for the target protein was incubated with Dynabeads Protein G (Invitrogen, Carlsbad, CA) in a tube for 10 min. Excess antibody was washed by placing the tube in a DynaMag™ magnet, as well as removing the supernatant. The antibody-coated beads were used for the following steps of IP). First, 107 of Raji cells were washed with PBS by centrifugation at 2500 × g for 10 min. Then, the supernatant was discarded. The cells were added into 1 ml of NP-40 with protease inhibitors and the mixture was gently shaken for 30 min on ice. Cell debris was removed by centrifugation at ∼14,000 × g for 15 min. The supernatant was transferred to a new tube. The antibody-coated beads were added into the cell lysates and incubated for 30 min at 4 °C. Excess component of the cells was washed by placing the tube in a DynaMag™ magnet, as well as removing the supernatant. The complexes of antigen-antibody-beads were re-suspended in 200 μL of PBS. Then, the complex was reduced and denatured and ran on the SDS-PAGE, and eventually the molecular weights of the antigen, and the heavy and light chains of antibody were identified.
Chemopreventive efficacy of stampidine in a murine breast cancer model
Published in Expert Opinion on Therapeutic Targets, 2020
Kazim Sahin, Cemal Orhan, Ibrahim Hanifi Ozercan, Mehmet Tuzcu, Birsen Elibol, Taha Koray Sahin, Ulkan Kilic, Sanjive Qazi, Fatih Mehmet Uckun
Mammary glands or tumor samples were harvested during necropsy and placed on dry ice and stored at −80ºC. Protein samples from tumor specimens as well as normal mammary glands of healthy control mice were used. Multiple samples of equal amounts of mammary gland or tumor tissue were pooled from four animals. To extract the proteins, pooled tissue samples from each group were homogenized in 1 ml ice-cold hypotonic buffer A containing 10 mM HEPES (pH 7.8), 10 mM KCl, 2 mM MgCl2, 1 mM DTT, 0.1 mM EDTA, and 0.1 mM phenylmethylsulfonyl-fluoride (PMSF). Eighty microliters of 10% Nonidet P-40 (NP-40) solution was added to the homogenates and the mixtures were centrifuged for 2 min at 14,000 g. The precipitates, containing nuclei, were washed once with 500 µl of buffer A plus 40 µl of 10% NP-40, centrifuged, resuspended in 200 µl of buffer C [50 mM HEPES (pH 7.8), 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 1 mM DTT, 0.1 mM PMSF, 20% glycerol], and centrifuged for 30 min at 14.800 g and the supernatant was transferred into fresh tubes. The tissue homogenates were collected for Western blot analyses of BRCA1, p21, Bcl-2, Bax, caspase-3, and β-actin.
Intracellular trafficking and cytotoxicity of a gelatine–doxorubicin conjugate in two breast cancer cell lines
Published in Journal of Drug Targeting, 2020
Mohammed M. Alvi, Rachel E. Nicoletto, Bayan A. Eshmawi, Hyun (Kate) Kim, Christopher R. Cammarata, Clyde M. Ofner
This procedure is modified from a previous report [40]. After detachment, cells were centrifuged and resuspended in 1 ml of ice-cold DPBS. Cells were counted, then lysed in hypotonic buffer and incubated over ice for 15 min. To this, 25 µl NP-40 (10%) was added and the nuclear fractions were pelleted. The nuclear fractions were suspended in 1 ml DPBS. DOX standards (10 µl) and a (10 µl) daunorubicin internal standard (25 µg/ml) in 0.03 M potassium phosphate buffer with 0.12 M NaCl (pH 4.8), 5 µl Triton-X 100 (5%), and 5 µl Proteinase K (10 mg/ml) were added to 200 µl samples. After mixing, samples were incubated for 1 h at 65 °C, followed by addition of 2.5 µl PMSF (10 mM in isopropanol), then incubated for 10 min at room temperature. To this, 5 µl of MgCl2 (0.4 M) and 10 µl of DNase I (1 mg/ml) were added and incubated for 30 min at 37 °C. The intra-nuclear DOX was extracted by equal volume (225 µl) of absolute methanol and 22.5 µl ZnS04 (400 mg/ml) followed by centrifugation. To a 30 µl aliquot of the supernatant, 50 µl ice-cold absolute methanol and 15 µl pure water were added and vortexed. Samples were stored at −80 °C for analysis within 72 h. The absence of DOX release during preparation of GDOX samples was confirmed by GDOX incubation for 30 min and analysis which produced negligible DOX nucleus accumulation.
Related Knowledge Centers
- Detergent
- Lipid Bilayer
- Nuclear Envelope
- Surfactant
- Cell Membrane
- Hydrophilic-Lipophilic Balance
- NONoxynol-9
- Triton X-100