China
Africa
Explore chapters and articles related to this topic
Calcium and Cytokinin in Mosses
Published in R. N. Chopra, Satish C. Bhatla, Bryophyte Development: Physiology and Biochemistry, 2019
Protein patterns and protein phosphorylation in chloronema and caulonema cells have been analyzed before and after cytokinin treatment (Figures 3 and 4). Protonemata were separated into chloronemata and caulonemata, preincubated in 32P (50 μCi/ml phosphate-free Laetsch’s medium) for 4 h ± 20 μM BA for 1 h. Homogenates were prepared from the labeled cells using a Dounce homogenizer and 440 μl of 10 mM phosphate buffer (pH 6.2, containing 5 mm EGTA, 100 mM sodium fluoride, and 50 μg/ml leupeptin) on ice. Cell debris was removed from the homogenate by centrifugation. Lipid was removed by Freon extraction and centrifugation. Protein was precipitated with 20% trichloroacetic acid, washed with acetone, and lyophilized. Sodium dodecyl sulfate Polyacrylamide gel electrophoresis (SDS PAGE) was performed according to Laemmli97 in a Bio-Rad® mini gel apparatus using 12% acrylamide gels. Gels were silver stained, autoradiographed, and analyzed using an LKB® laser densitometer. No visible changes in the synthesis of proteins could be observed on these one-dimensional gels. However, a 50-kDa and a 47-kDa protein were phosphorylated only in caulonema cells treated with B A.
Analysis of Normal and Neoplastic Tissue NHC Proteins by High-Resolution Two-Dimensional Gradient Electrophoresis and Silver Staining
Published in Isaac Bekhor, Carol J. Mirell, C. C. Liew, Progress in Nonhistone Protein Research, 1985
Normal tissues and/or tumors are removed from fasted animals, weighed and washed in ice-cold physiologic saline containing phenylmethylsulfonyl fluoride (PMSF). Washed tissues are minced and homogenized in 10 volumes of 0.25 M STM (0.25 M sucrose-0.01 M Tris-HCl (pH 7.4), 0.025 M MgCl2) in 1.0 mM PMSF. This and all subsequent solutions and buffers contain 1.0 mM PMSF and all steps are performed at 4°C. After filtering homogenates through four layers of cheesecloth, crude nuclei are pelleted by centrifugation at 5000 rpm for 10 min (Sorval SS34 head or comparable rotor). Supernatants are collected and set aside for later cytosolic protein isolation. Pelleted nuclei are resuspended in 0.25 M STM, an aliquot examined by light microscopy to determine that all whole cells have been disrupted, and then two volumes of 2.3 M sucrose, 0.5 M KCl, 0.025 M MgCl2 in 0.01 M Tris-HCl (pH 7.4) containing 0.1% Triton® X-100 are added. Nuclei are isolated by centrifugation at 30,000 rpm in a fixed angle 30 rotor (Beckman) for 1 hr. Nuclei are resuspended and washed three times in 10 mM Tris (pH 8.0)-1.0 mM PMSF. Phase contrast and electron microscopic analysis of Triton® X-100-prepared nuclei confirm the absence of cytoplasmic tags and the removal of the outer nuclear membrane. Histones are extracted with dilute mineral acids, recovered and acid-urea gels run to analyze for presence of electrophoretic sub-bands, a highly sensitive assay for chromosomal protein proteolysis.81 Nuclei suspended in Tris-buffer are lysed by 8 strokes in a tight-fitting Dounce homogenizer (“B” pestle). Then, 7 mℓ of lysed chromatin solution are layered over sucrose step gradients of 15 mℓ each of 1.6 M sucrose and 15 mℓ of 1.3 M sucrose (both in 10 mM Tris-PMSF at pH 8.0) and sedimented at 27,000 rpm for 90 min (SW27 rotor, Beckman). Chromatin pellets are resuspended in 0.01 M Tris HCl pH 8.0, lightly homogenized and layered a second time over step gradients composed of 3 mℓ each of the 1.6 and 1.3 M sucrose. Gradients are centrifuged at 41,000 rpm for 60 min (SW41, Beckman). Pellets are washed gently three times in 0.01 M Tris-HCl (pH 8.0) and then dialyzed overnight against 10 mM Tris-HCl (pH 8.0)-PMSF, quick-frozen in a dry ice-acetone bath, and stored at −80°C. To ensure that chromatin preparations are free of cytosol contamination, aliquots of FH]-labeled cytoplasm prepared from livers of male 175-g Sprague-Dawley rats exposed to 0.1 Ci/g body weight of [3H]-labeled amino acid mix are used as part of the homogenization medium. Post-nuclear supernatants (crude nuclear isolation step) are combined and centrifuged at 60,000 rpm in a 60Ti rotor (Beckman) for 1 hr. This procedure pellets mitochondria, ribosomes, and particulate components. Cytosolic supernatants are combined, dialyzed against H2O containing 1.0 mM PMSF, shell frozen, lyophilized, and stored at −80°C. Yields of DNA-chromatin with these procedures typically are 0.8 to 1.0 mg/g of starting wet weight, yield from PHCs reflecting that from necrosis-free tissue.
Identification of protease serine S1 family member 53 as a mitochondrial protein in murine islet beta cells
Published in Islets, 2022
Noriko Mizusawa, Nagakatsu Harada, Takeo Iwata, Izumi Ohigashi, Mitsuo Itakura, Katsuhiko Yoshimoto
pcDNA3.1/myc-His-Prss53-expressing COS-7 cells were washed thrice with PBS and scraped into a homogenizing buffer (10 mM Tris-HCl [pH 7.4], 0.25 M sucrose, 2 mM EDTA, and 1 mM PMSF). Next, the cells were homogenized using a Dounce homogenizer with 12 strokes of an A-type pestle. The homogenate was centrifuged at 1000 × g for 10 min to remove the nuclei and large cell fragments. The post-nuclear supernatant (PNS) was subjected to centrifugation at 105,000 × g for 60 min to obtain a membrane fraction (pellet; Mm) and a cytosolic fraction (supernatant; Cy). Additionally, the PNS was centrifuged at 10,000 × g for 12 min. The resulting supernatant was centrifuged at 105,000 × g for 60 min to obtain the Org fraction as a pellet. The Mm and Org fractions were resuspended in a homogenizing buffer containing 0.5% Triton X-100, and the homogenate was subjected to western blotting analysis. The Org fraction was used for further analysis.
Differential expression and transcription factor binding associated with genotype at a pharmacogenetic variant in OPRD1
Published in The American Journal of Drug and Alcohol Abuse, 2021
Richard C. Crist, Gabriella Arauco-Shapiro, Alexander Zhang, Benjamin C. Reiner, Wade H. Berrettini, Glenn A. Doyle
Double-stranded DNA probes (15bp) for both alleles of rs678849 were synthesized, annealed, and purified by Integrated DNA Technologies (C allele sense sequence: 5ʹ-TCAAAAGCACCTGCT-3ʹ; T allele sense sequence: 5ʹ-TCAAAAGTACCTGCT-3ʹ). The fluorophore Cy3 was conjugated to the 3ʹ end of the sense strands for each probe. Unlabeled versions of the C and T allele probes were also produced, as well as an unlabeled competitor probe containing the murine Oct1 consensus sequence (TCGAATGCAAATCAC). BE(2)C nuclear lysate was obtained using a Nuclear Extraction Kit (AbCam). Postmortem medial prefrontal cortex tissue was received from NIH NeuroBioBank at the University of Maryland. Tissue was homogenized in a Dounce homogenizer and nuclear lysate was again obtained using the Nuclear Extraction Kit. Double-stranded T probe (2 pmol) was incubated with 20 μg nuclear lysate for 25 min at room temperature in 20 μL modified 1X Cold Spring Harbor EMSA binding buffer (30 mM NaCl, 5 mM HEPES-KOH (pH 7.6), 1.2 mM DTT, 20 μM EDTA, 83.3 nM poly-dIdC). Double-stranded C probe incubation was performed as above but using dephosphorylated lysate to remove a prominent nonspecific band (Figure 2A). Dephosphorylated lysate was generated by incubating BE(2)C or medial prefrontal cortex nuclear lysate with 1 U calf intestinal alkaline phosphatase per 80ug lysate (NEB) for 30 minutes at 37°.
Higher Glucose Enhances Breast Cancer Cell Aggressiveness
Published in Nutrition and Cancer, 2020
Julianna M. Santos, Fazle Hussain
Breast cancer cells (MCF-7 and MB231) were trypsinized and centrifuged for 5 min at 1,000 rpm, washed three times with DPBS (Corning Cellgro, 21-031-CV), and resuspended in 3 ml of homogenizer buffer containing 1.3 M sucrose (Sigma-Aldrich, S7903i), 1 mM MgCl2 (Sigma-Aldrich, M4880), and 10 mM potassium phosphate buffer pH 6.8 (Sigma-Aldrich, P5379 and Fisher Chemical Fisher Scientific, BP363-500), supplemented with a protease inhibitor cocktail (Thermo Fisher Scientific, 88265) at a concentration of one tablet per 50 ml. Cells were homogenized 20 times in a Teflon/glass dounce homogenizer (Sigma Aldrich). The suspension was stored as a homogenate fraction (containing all cellular fractions including plasma membrane, cytosol, and nucleus) at −80 °C. The protein concentration was determined with a spectrophotometer/fluorometer (DeNovix, DS11 FX+) using nano drop at 280 nm prior to performing western blots.