Cell lysate separation

Figure 13.2-2. Repeat ultraviolet-traces (280nm) recorded for a representative yeast whole cell lysate separated by lEX-HPLC showing the reproducibility of the procedure.

FIGURE 33.3 (See color insert following page 810.) (a) Microfluidic PMMA chip for 2-D lEF/SDS-CGE protein separations and (b) pseudo-gel image resulting from a yeast cell lysate separation. 

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.

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... 

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. 

Wall, D. B. Lubman, D. M. Flynn, S. J. Rapid profiling of induced proteins in bacteria using MALDI-TOF mass spectrometric detection of nonporous RP HPLC-separated whole cell lysates. Anal. Chem. 1999, 71,3894-3900. 

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. 

Figure 10.1 Experimental schemes for microarray analysis. All experimental schemes start with a separation step of the cell lysate by velocity sedimentation in a sucrose gradient (top scheme). Collection of the desired fractions is assisted by a continuous ultraviolet (UV) reading of the gradient (an example of such UV reading is shown in each section). This allows determination of the sedimentation position of the 40S, 60S, 80S, and polyribosomal complexes (2,3, and more).Three general ways for fraction collection and analysis are presented (sections A, B, and C) (A) Collection of two fractions (free and polysomes) and direct comparison between them, with the free mRNA fraction labeled with green dye and the polysome fraction labeled with red dye. (B) Collection of two fractions and indirect comparison between them by utilizing an unfractionated reference RNA. (C) Collection of multiple fractions (four in this case), where each fraction is compared to an unfractionated reference sample. The blue arrows indicate the addition of spike-in RNA to each fraction and to the reference RNA.

Figure 4.14. Separation of cell lysates containing pharmaceutical products, employing large-capacity centrifuges that are configured with tubular bowl (A), multi-chamber bowl (B), disc stack bowl (C), or decanter (scroll) bowl (D).

When the C-terminal domain is expressed as a separate polypeptide, IN213-288 can be purified from the initial soluble fraction from cell lysates [33]. This small protein fragment, therefore, was an attractive target for structure determination. 

Besides the enzymatic incubation in the reaction mixture, all procedures are carried out at 4°C. GTPCH activity is assayed by measuring the neopterin produced upon enzymatic incubation at 37°C for 60 min in a final volume of 0.1 ml in the dark (due to light sensitivity of pterins), followed by chemical oxidation and dephosphorylation. Two separate blanks are prepared, a blank reaction with cell lysate that is immediately oxidized to detect the neopterin that was present in the lysate, and a blank reaction without cell lysate to detect the neopterin that is generated from the incubation (substrate) buffer. The sum of both blanks is later subtracted from the value of the incubation reaction to determine the enzymatically produced neopterin. 

The cells and cell lysates (fragments of disrupted cells) can be separated from the soluble components by using microfiltration (Chapter 8) with membranes. This separation method offers following advantages ... 

The lysate was obtained from E. coli cells which were pre-transformed with a plasmid (pSTB7) expressing tryptophan synthase from Salmonella enterica. This E. coli strain is commercially available (ATCC 37845). Reported yields on tryptophans ranged from 3% to 63%, but, in several cases, could be significantly enhanced up to 100% using the E. coli lysate, separated from the reactant by a dialysis tube. 

Figure 2 SDS-PAGE analysis of purified HRV14 2A and 3C proteases. Protein samples (—1-2 (ig) were separated by electrophoresis on 16% gels and then stained with Coomas-sie blue. Lanes 1-4 represent the purification ofHRV14 2A protein. Lanes 1, transformed cell lysate 2, urea-solubilized inclusion bodies 3 and 4, 2A protease preparations after Mono Q and Superdex-75 columns, respectively. Lane 5 represents the purified HRV14 3C protease.

Phenolic extraction of cell lysates is one of the oldest techniques in DNA preparation. Examples of these have been presented in Chapters 6 and 7. Single cells in suspension are lysed with a detergent, and a proteinase enzyme is used to break down the protein molecules. Non-nucleic acid components are then extracted into an organic (phenol-chloroform) solvent, leaving nucleic acids in the aqueous layer. Two volumes of isopropanol are added to the isolated aqueous phase to precipitate the high-molecular-weight nucleic acids as a white mass. These are then treated with DNase-free ribonuclease (RNase) to remove the RNA. This is followed by a second treatment with proteinase, phenol extraction, and isopropanol precipitation. After precipitation, the DNA is separated from the isopropanol by... 

Separation of proteins extracted from Jurkat cell lysates was achieved on a microchannel on a PMMA chip. Pressurization has been used prior to CE to improve resolution, as described in section 6.2.2. An additional merit is that transient size separation occurred during pressurization. During subsequent CE separation, IEF occurred in the microchannel. This combination of transient size separation and IEF improved resolution, which is akin to 2D separation [619]. 

Fig. 9 Optimization of sample injection volume. Various injection volumes (VL) of spiked cell lysate samples were investigated to optimize VL. Lysates were spiked with 250 pg/mL of paclitaxel and extracted by selective SPE. The VL was (a) 0.1 pL (b) 0.5 pL (c) 2.0 pL (d) 8.0 pL. A 15 cm x 0.5 mm I.D. capillary column was used for separation the manufacturer-recommended Vnj for the column was 0.1-0.2 pL (Reproduced with permission from Elsevier)...

Fig. 1.6 Effect of NAC and DPI on ERK 1/2 phosphorylation in vascular smooth muscle cells (VSMC) from 12 week-old SHR and WKY rats. Confluent VSMC from SHR and WKY rats were treated with 20 mM IV-acetyl-L-cysteine (NAC) or 10/r.M diphenyleneiodonium (DPI) for 24 hours at 37 °C. Cell lysates were immunoblotted by phospho-spccif-Tyr204-liRKI/2 antibodies as shown in the top panel. Blots were also analyzed for total ERK1/2 (bottom panel). The blots are representative of three separate experiments. Detection of p-ERKl/2 and total ERK1/2 was performed with chemiluminescence Western blotting detection reagents. WKY levels were taken as 100%. Values are mean S.E.M. of three separate experiments. P < 0.01, p < 0.001 versus WKY and < 0.001 versus SHR. Reproduced with permission from Lappas et al. (2005).

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