Cell lysate

Cell lysates from mutant strains of X. campestris were incubated with radiolabelled UDP[14C] glucose or GDP[,4C] mannose, the other sugar nudeotide substrates being unlabelled. The reaction mixture was then divided into lipid and soluble fractions. Where would you expect the radiolabel to be found and what produd, if any, would you expert from strains with defidendes in the following genes ... 

The previous ELP fusions all are examples of protein purification in which the ELP is covalently connected to the protein of choice. This approach is suitable for the purification of recombinant proteins that are expressed to high levels, but at very low concentrations of ELP the recovery becomes limited. Therefore this approach is not applicable for proteins expressed at micrograms per liter of bacterial culture, such as toxic proteins and complex multidomain proteins. An adjusted variant of ITC was designed to solve this problem. This variant makes use of coaggregation of free ELPs with ELP fusion proteins. In this coaggregation process, an excess of free ELP is added to a cell lysate to induce the phase transition at low concentrations of... 

Figure 2. PemB cellular localisation. (A) Fractionation of E. chrysanthemi cells by spheroplasting. Lane 1, culture supernatant lane 2, total cell lysate lane 3, periplasmic fraction lane 4, crude membrane fraction lane 5, cytoplasmic fraction. (B) Detergent extraction of PemB from E. chrysanthemi A837 cell envelopes. Lane 1 crude envelope fraction lane 2 Triton-soluble fraction lane 3 Triton-insoluble fraction lane 4 Sarkosyl-soluble fraction lane 5 Sarkosyl-insoluble fraction.

Figure 4. Purification of PemB from E. coli K38 pGPl-2/pPME6-5 cells. Proteins were separated by urea-SDS-PAGE. Lane 1, induced cell lysate lane 2, soluble protein fraction from induced cells lane 3, membrane fraction from non-induced cells lane 4, membrane fraction from induced cells lane 5, membrane proteins not extracted by Triton X-100 lane 6, membrane proteins extracted by Triton X-100 lane 7, PemB purified by preparative electrophoresis. The molecular weight standard positions are indicated.

Collier et al. (1990) extended their studies relating to oxidative stress and diabetes by demonstrating that the levels of several free-radical scavengers (red cell superoxide dismutase, plasma thiols) were significantly reduced in 22 type 2 diabetic patients (mean age 53 years) in comparison with 15 control subjects (mean age 51 years). No significant diflFerences in red cell lysate thiols or... 

Protein affinity chromatography can be used for the separation of an individual compound, or a group of structurally similar compounds from crude-reaction mixtures, fermentation broths, or cell lysates by exploiting very specific and well-defined molecular interactions... 

Saborio GP, Soto C, Kascsak RJ, Levy E, Kascsak R, Harris DA, Frangione B. Cell-lysate conversion of prion protein into its protease-resistant isoform suggests the participation of a cellular chaperone. Biochem Biophys Res Commun 1999 258 470-475. 

Prokaryotic cells express hundreds to thousands of proteins while higher eukaryotes express thousands to tens of thousands of proteins at any given time. If these proteins are to be individually identified and characterized, they must be efficiently fractionated. One-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) has typically been use to study protein mixtures of <100 proteins. Onedimensional electrophoresis is useful because nearly all proteins are soluble in SDS, molecules ranging from approximately 10,000 to 300,000 molecular weight can be resolved, and extremely basic or acidic proteins can be visualized. The major disadvantage to one-dimensional gels is that they are not suitable for complex mixtures such as proteins from whole cell lysates. 

In principle, the use of addressable, pooled GST fusion proteins could be used to identify proteins associated with any biochemical activity, assuming that the fusion protein is soluble, folded and functional. The method has the additional advantage that, once the GST fusion clones are constructed, it is a rapid technique. The authors state that only two weeks are required to purify the 64 pools and the assays can be accomplished in a day (Martzen et al., 1999). In addition, the method is sensitive because only 96 recombinant proteins are assayed at one time in contrast to the use of cell lysates where thousands of proteins are present. This leads to a much higher concentration of each protein, which greatly facilitates detection of a biochemical activity (Martzen et al., 1999). 

Chong, B. E. Wall, D. B. Lubman, D. M. Flynn, S. J. Rapid profiling of E. coli proteins up to 500 kDa from whole cell lysates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Comm. Mass Spectrom. 1997,11,1900-1908. 

Xiang, F. Anderson, G. A. Veenstra, T. D. Lipton, M. S. Smith, R. D. Characterization of microorganisms and biomarker development from global ESI-MS/MS analyses of cell lysates. Anal. Chem. 2000, 72, 2475-2481. 

Zhu, Y. Lubman, D. M. Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations. Electrophoresis 2004, 25, 949-958. 

HILIC RPLC UV, MS Protein mix, cell lysates 8-port Murphy (2001)... 

CF RPLC MS Breast cancer cell lysates Fraction collector Chong et al. (2001)... 

Chong, B.E., Yan, F., Lubman, D.M., Miller, F.R. (2001). Chromatofocusing nonporous reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry of proteins from human breast cancer whole cell lysates a novel two-dimensional liquid chromatography/mass spectrometry method. Rapid Com-mun. Mass Spectrom. 15, 291-296. 

This instrument was used to analyze mixtures of intact proteins, including protein standards and a cell lysate of the bacterium Escherichia coli. A UV... 

ProteoVue pf/hydroptiobicity map for an E.coli 0157 H7 whole cell lysate... 

FIGURE 10.2 2D map of a whole cell lysate (top) along with an illustration of the reproducibility of the p / versus hydrophobicity profiling technique (bottom) using the Beckman PF2D automated instrument and software. (See color plate.)... 

Buchanan, N. S., Hamler, R. L., Leopold, P. E., Miller, F. R., Lubman, D. M. (2005). Mass mapping of cancer cell lysates using two-dimensional liquid separations, electrospray-time of flight-mass spectrometry, and automated data processing. Electrophoresis 26(1), 248-256. Buick, R. N., Pullano, R., Trent, J. M. (1985). Comparative properties of 5 human ovarian adenocarcinoma celllines. Cancer Res. 45(8), 3668-3676. 

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