To purify the protein in the cell lysate from lab 1 through nickel affinity chromatography. Protein purification should result in only one type of protein ideally‚ which is the protein of interest‚ wt-DHFR and mut-DHFR in this case. A pure protein allows for further analysis on the protein to be conducted‚ such as its concentration (Bradford assay)‚ its molecular weight‚ and its biological activity. 2. Overview of experiments Buffer Preparation Add the liquid sodium phosphate‚ solid sodium chloride
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1.5.2.5 Detectors The detectors used in UPLC should be able to handle very fast scanning methods because half-height peak widths of less than one second are usually obtained with columns packed with 1.7 µm particles. The detector must be able to give high sampling rate adequate to capture enough data points across the peak for an accurate and reproducible integration of analyte peak. The dispersion (volume) of the detector flow cell must be minimal to maintain separation efficiency. Conceptually
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EXPERIMENT 4 ANALYSIS OF HYDROCARBON IN COMMON FUELS BY SOLID-PHASE MICROEXTRACTION (SPME) AND GAS CHROMATOGRAPHY-MASS SPECTROMETRY (GC-MS) Abstract In the analysis the solid-phase microextraction (SPME) and capillary gas chromatography/mass spectrometry (GC/MS) was developed for the identification of volatile compounds (hydrocarbon) in fuel. The samples was used is (kerosene‚ diesel‚ thinner and petrol) and one unknown. After the analyte was extracted by SPME in 20min‚ it directly injected
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bp). SDS-PAGE analysis of auto-induced positive clone cell lysate revealed the expression of the cfp-wzb fusion protein (experimental: 44.3 kDa). Using a hexahistidine tag‚ successful fusion protein purification was achieved via nickel affinity chromatography and confirmed by SDS-PAGE. Finally‚ para-nitrophenyl phosphate assay of tyrosine phosphatase activity allowed for determination of wzb kinetic properties such as Km (6.34 mM) and Vmax (0.0644 µmol/min/mg). Gaining an experimental competency in
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of the sample (as analyzed by 1H and 13C NMR) was poor. There are many factors that may have caused poor purity‚ of which include reaction incompletion during exposure to microwave radiation‚ poor mixing‚ and poor product isolation during column chromatography. As seen in the 1H NMR spectrum‚ impurity peaks (>10%) corresponding to methyl (triphenylphosphoranylidene) acetate‚ acetone‚ ethyl acetate‚ and hexane were identified. The phosphonium ylide peak indicates that the reaction did not go to completion
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GM‚ Kriz GS‚ Engel RG. (2011). A Small Scale Approach to Organic Laboratory Techniques. Location: Unknown. Publisher: Mary Finch (August 10‚ 2006). Retrieved November 13‚ 2012‚ from http://www.chem.ucla.edu/~bacher/General/30BL/gc/theory.html Chromatography. Introductory Theory. Retrieved November 13‚ 2012‚ from http://teaching.shu.ac.uk/hwb/chemistry/tutorials/chrom/chrom1.htm
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velmpharm24@gmail.com A precise‚ accurate and rapid RP-HPLC method has been developed and validated for the simultaneous estimation of Tamsulosin Hydrochloride and Finasteride in tablet dosage form. Chromatography was carried on RP – 18e Hiber RT (250 × 4.6) column using Water:Methanol (30:70%) v/v as mobile phase at a flow rate 0.7 ml/min and the effluent was monitored at 225 nm. The retention times of Tamsulosin Hydrochloride and Finasteride were 5.4 and 15.4 minutes respectively
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filtration GEL FILTRATION OF PROTIENS Aim: The aim of this experiment is to identify proteins from a complex mixture using the gel filtration technique also known as size exclusion chromatography. This technique is widely used by biochemists when proteins larger than the pores are excluded from the column and the smaller molecules elute last and then collected in test tubes for examination by spectroscopic techniques. The red/brown proteins‚ in particular‚ will be observed closely as they
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Purpose The purpose of this experiment is to exemplify how differences in molecular weight allow separation of polymers from their monomers. Methods of dialysis and gel filtration chromatography will be used to separate a glucose monomer from a starch polymer. Colorimetric glucose oxidase assay will be used to monitor the presence of glucose and a colorimetric iodine assay will be used to monitor the presence of starch in prepared solutions
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IDENTIFICATION OF UNKNOWNS BY TLC AND MP IN COMBINATION Introduction: Thin layer chromatography (TLC) is one of the most valuable techniques in organic chemistry. This is a best method of separating and identifying mixtures of two or more compounds. The separation is accomplished by the distribution of the mixture between two phases: one that is stationary and one that is moving or mobile. Chromatography works on the principle that different compounds will have different solubilities and adsorption
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