Lovastatin Research Paper

Superior Essays
INTRODUCTION Lovastatin is a statin drug that decreases the total cholesterol level in patients diagnosed with hypercholesterolemia. The drug aims to prevent serious cardiovascular diseases such as heart attack due to blockage of arteries. Lovastatin acts as a competitive inhibitor for the HMG-CoA reductase enzyme in catalyzing the conversion of HMG-CoA to mevalonate, (Alberts, 1998). The drug is also hydrolyzes in vivo to active form β-hydroxyl acid, (Katz, 2005) and was recently researched on its chemotherapeutic effects on certain types of cancer. Lovastatin also reduces proteasome activity causing an accumulation of the cyclin-dependent kinase inhibitors in breast cancer, (Rao et al. 1999). The purpose of this experiment is to study quantitatively …show more content…
The methodology of the experiment was based on the stable isotope labeling by amino acids in cell culture (SILAC) experiment whereby the Iscove’s modified minimal medium (IMEM) was custom prepared based on the ATCC formula without L-lysine or L-arginine. However, the complete light and heavy IMEM media was prepared in the addition of light and heavy lysine and arginine along in dialyzed FBS. The collected HL-60 cells were cultured in heavy IMEM medium for five cell doublings to achieve a complete isotope incorporation. The cultured medium was supplemented with 10% of fetal bovine serum and penicillin as antibiotics. The cultured cells were kept in a humidified atmosphere in presence of 5% CO2 at 370C. The medium used were renewed every two to three days depending on the cell density. Moreover, the SILAC experiment was based on two types which were forward SILAC and reverse SILAC. In forward SILAC, the HL-60 cells were cultured in light medium and treated with 10µM of lovastatin for 24 hours and the cells cultured in the heavy medium was used as an untreated control. In the reverse SILAC experiment, the cells were cultured in the heavy medium and were treated with lovastatin and the cells cultured in the light medium was used as a treated …show more content…
The proteins were in-gel digested with trypsin overnight forming peptides. The peptides were extracted with 5% acetic acid in H2O and 5% acetic acid in CH3CN/ H2O at a 1:1 ratio, dried and stored at -200C for further analysis.
B) Protein Identification A high performance liquid chromatography separation was used in presence of homemade trapping column and separation column. Both the column were packed with ReproSil-Pur C18-AQ resin. The determined peptide mixture was loaded into the trapping column with a solvent mixture of 0.1% formic acid at a flow rate of 4.0µL/min. The separation of the peptides were then carried out at 120 minutes in 2-4% acetonitrile and 0.1% formic acid at a flow rate of 300nL/min. A LC-MS/MS analysis was performed on an LTQ-Orbitrap Velos mass spectrometer and operated in a positive-ion mode at a voltage of 1.8kV. The twenty most abundant ions in mass spectrometry at a threshold above 500 counts were determined for fragmentation by collision-induced dissociation at a 35% collision energy.

4) Data analysis and verification
A) Target

Related Documents

  • Great Essays

    To attain conjugation of PEG-EGFR peptide on PF0-HNC, first the carboxyl group located on PEG-EGFR peptide was activated by the addition of 20% molar excess of N-hydroxysulfo-succinimide (NHS) and EDC followed by the addition of this carboxyl activated peptide to PF0-HNC to form PF0-HNC-E. The reaction was continued for 6 hr followed by the addition of 1 M glycine to the reaction mixture to quench the activated carboxyl group and terminate the reaction [43, 44]. The resultant conjugate was purified by dialysis using dialysis membrane (molecular weight cut off 15kD) against 500 ml of PBS for 4 hr to remove the free PEG-EGFR peptide, NHS, and EDC. The carboxyl-PEG-EGFR peptide (American Peptides, USA) was…

    • 2193 Words
    • 9 Pages
    Great Essays
  • Superior Essays

    To prepare the standard calibration samples, 50µL of standard solution were added to 100µL of liver homogenate. The mixture was vortex-mixed for 10 min; followed by centrifugation at 4000 rpm for 15 min. 20µL of the supernatant was taken, then filtered through 0.22µm size syringe filter and injected in HPLC for analysis. The final standard Raloxifene Hydrochloride concentrations were 30-150µg/mL. [6] 3.8 ANALYTICAL INTERFERENCE…

    • 777 Words
    • 4 Pages
    Superior Essays
  • Improved Essays

    Ddp Chemotherapy

    • 1238 Words
    • 5 Pages

    An area of interest for this study was to inhibit tumor angiogenesis, crucial in tumor development and growth. The tests in this study were conducted on NCI-H446 human small-cell lung cancer cells in vitro and in vivo in athymic nude mice. This new treatment method downregulated survivin but intensified tumor suppression in NCI-H446 SCLC cells (Li et al.,…

    • 1238 Words
    • 5 Pages
    Improved Essays
  • Improved Essays

    After 2 washings by centrifugation at 1200 rpm for 5 minutes in PBS, cells were plated in 75-cm2 tissue-culture flasks with concentrations of 0.3 to 0.4 × 106 cells/cm2 in the DEMEM medium supplemented with 15% fetal calf serum. Cells were incubated in a humidified 5% CO2 at 37 °C. Four days following primary culture initiation, the culture mediums were collected, centrifuged and the cell…

    • 796 Words
    • 4 Pages
    Improved Essays
  • Improved Essays

    Centrifuged the test tubes at 4000 rpm for 10 minutes and the absorbance of the upper layer were measured at 532 nm. Standard malondialdehyde was taken as the standard and was processed in the same manner. The level of lipid peroxide was expressed as µmoles of MDA formed /g wet…

    • 801 Words
    • 4 Pages
    Improved Essays
  • Improved Essays

    About 5 mg of the sample was suspended in 50µl of methoxylamine hydrochloride solution in GC grade pyridine (20 mg ml-1). The mixture was shaken for 2 hr. at 370C before adding 70 µl of MSTFA. Shaking was continued for another 30 min. Analysis of lipid content by GC–MS was performed using Thermo Trace GC Ultra coupled with Thermo fisher DSQ II mass spectrometers.…

    • 1032 Words
    • 5 Pages
    Improved Essays
  • Great Essays

    Transfer all medium from each well to 1.5 ml tubes. Centrifuge at high speed 10000g for 1 min in Eppendorf tube. Transfer supernatant to 1.5 ml tube and snap frozen in liquid nitrogen then kept at -80 °C until hormonal analysis. Trypsinization were used for harvesting live cell, 500 µl of pre-warmed trypsin-EDTA were added to each well and incubated at 37.5 °C for 5 min. Then dislodged cells from well by pipetting and to stop trypsin effect, 100 µl of heated inactivated fatal calve serum (HI FCS) were added to each well and mixed well.…

    • 984 Words
    • 4 Pages
    Great Essays
  • Decent Essays

    Then, a solution of 0.5 M Zn(NO3)2.6H2O was added dropwise under constant stirring. For collection of core ZnO, half portion of the material was filtered and washed several times with deionized water and ethanol. The washed samples were dried at 650 C in the oven for 1 hour. The shell CdS nanoparticles were prepared by simultaneous addition of Cadmium Acetate solutions and Sodium Sulfide (Na2S) solutions. The synthesis was carried out at room temperature.…

    • 748 Words
    • 3 Pages
    Decent Essays
  • Improved Essays

    Psg Research Paper

    • 1197 Words
    • 5 Pages

    Methods and materials Cell culture medium Making cell culture medium, requires two processes, this includes the medium that has PSG and other without the PSG. 15 ml of 10% FBS was added into a culture flask with 1.5 ml 100x hippos. The culture flask was filled with DMEM up to 150 ml and this will be the medium without the PSG and this process completed the medium without the PSG. The same procedure was followed for the PSG medium, 1.5 ml of PSG was added and that completed the medium with PSG. Bacterial culture plates for NFkb and GFB 3.5 g of 7 g/250 ml agar and 6.5 g of 25 g/100 ml LB was added to a 500 ml flask.…

    • 1197 Words
    • 5 Pages
    Improved Essays
  • Improved Essays

    An NC membrane provides sites for immobilization of capturing antibodies. The LFA strips were optimized and prepared as mentioned by Posthuma -Trumpie et al. (2008) with some modifications. Briefly, the SSAbs and anti horse antibodies were immobilized on plastic backed NC membrane (10 µm) at test and control lines respectively, with 1 µl/cm using TLC Spotter (Camag Linomat IV, Camag Berlin, Germany). These membranes were dried at 37 o C for 1 hr, blocked with PBS containing 1% BSA for 30 min, followed by washing two times with PBS and drying at 37 o C for 2 hrs.…

    • 1742 Words
    • 7 Pages
    Improved Essays