Characterisation of GST-GRP1PH
GST-GRP1PH was characterized using SDS–PAGE with sensitive Coomassie stain followed by automated in-gel digestion and LC-MS/MS analysis (results not shown) [64]. An 85% peptide coverage of the PH domain was obtained using MS/MS analysis (results not shown).
GST-GRP1PH binding specificity toward PI(3,4,5)P3 was analysed using both overlay phosphoinositide assay and Biosensor analysis (Figure 2). GST-GRP1PH was found to recognize specifically immobilized PI(3,4,5)P3 when compared to immobilized PI(4,5)P2 (Figure 2A) and 2B). A standard curve generated by plotting the average intensity of the dot blot versus the amount of immobilized PI(3,4,5)P3 showed that approximately 4ng (3.3pmoles) of PI(3,4,5)P3 could be detected in this assay.
Binding specificity was also characterized using biosensor analysis. GST-GRP1-PH was injected at various concentrations (1.5µM, 750nM, 375nM, 188nM, 94nM) over immobilized PC/PE/PI(4,5)P2 and PC/PE/PI(3,4,5)P3 liposomes (Figure 2C and 2D). GST-GRP1PH was observed to bind specifically to immobilized PC/PE/PI(3,4,5)P3 liposomes (Figure 2C) as compared to immobilized PC/PE/PI(4,5)P2 PC/PE (Figure 2D).
Kinetic constants were extracted from the biosensor curves from global analysis using 1/1 Langmuir binding with mass transfer. Analysis of the interaction between GST-GRP1PH and immobilized PI(3,4,5)P3 gave an apparent association rate (ka) of 4.5 x 104M-1s-1 and apparent dissociation rate (kd) of 1.6 x 10-3s-1 resulting in an equilibrium dissociation constant (KD) of 35.5nM.
Fluorescent imaging of GST-GRP1PH in resting and EGF stimulated HEK293T, LIM1215 and LIM2550 cells
Confocal microscopy was used to analyse PI(3,4,5)P3 localisation using GST-GRP1PH in resting and...
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...nhibition on the EGF-dependent redistribution of GST-GRP1PH. LY294002, a specific and potent inhibitor of PI3K, was used in these experiments (Figure 6). HEK293T, LIM1215 and LIM2550 cells were incubated in 1, 3.12, 6.25, 12.5, 25, 50 and 100µM inhibitor for an hour before stimulation with EGF for 5min. . LY294002 inhibited the formation of PI(3,4,5)P3 at the plasma membrane following EGF stimulation in a concentration dependent manner, as detected by fluorescence confocal imaging (Figure 6). The membrane/cytoplasm fluorescence ratio was calculated for each inhibitor concentration and used to generate inhibitor response graphs (Figure 7). One phase decay non-linear regression analysis was performed for each cell line and the IC50 value calculated (Figure 7). The IC50 values for the HEK293T, LIM1215 and LIM2550 cell lines were 5.7µM, 1.7µM and 8.7µM respectively.
Ligation of EGFP into pET41a(+) vector transformed into E. coli cells followed by PCR amplification of extracted DNA plasmid for success evaluation along with gel electrophoresis at each step.
Craig, D. Q. (2002). Pharmaceutical Applications of Micro-Thermal Analysis. Journal of Pharmaceutical Science, 91(5), 1201-1213.
Co-IP is the most commonly used methods to verify protein-protein interactions (Berggård et al., 2007). Antibodies that are specific to the bait complexes are used to capture the bait complexes in a cell lysate shown in Fig. 1. The antibody is immobilized on Protein A/G, which is covalently bound to the agarose beads. Since the antibody is specific to only the bait complex, the antibody will not bind to other proteins found in the cell lysate, and hence, these proteins will be wash off. The antibody-bait compl...
The structures of liposomes are spherical and are usually between 15nm and 1000 nm in diameter. They are able to target the ligands that are attached to their surface to direct them to the appropriate sites wi...
Acknowledgements. Special thanks go to the Department of Chemistry and Chemical Biology at IUPUI, Dr. Ryan E. Denton, Professor and Dan Preston, TA.
...des dissolving of 100mg of PC into 15 ml ethanol and then this solution mixture is added drop-wise into a Vitamin C solution. Continuous stirring is required. The conditions like low temperature and moisture content can be achieved. The organic solvent is then evaporated and by maintaining pH at 7.4 of the phosphate buffer solution (PBS), the solvent traces are removed. The Liposome dispersion is then stored under vacuum overnight. The liposome size can be downsized by sonication. Liposome characterisation i.e. size and surface structure can be observed using cryo-transmission electron microscopy (cryo-TEM) (27).
CP consists of a single domain with high α-helical content [4]. The N-terminal part this domain is surface exposed whereas the C-terminal region buried in the virion. Several experiments indicate the CP is an O-glycoprotein. Equal amounts of galactose and fructose residues are O-linked to an acetylated serine residue at the N-terminal region [2]. This mediates the formation of a structured...
Once binding has occurred, a cascade of signalling reactions will initiate, with Rho guanosine-5'-triphosphate (Rho GTPases) such as rho-asso...
...lications in the future. This is due to the fact that this method has become rough, not complicated and it can be performed in a conventional way without being mandatory the investigation into depth for every application (Tetala and van Beek, 2010). New forms are going to be operated in order to recognize bacteria and also aptamers are going to be used more often. Moreover, the investigation of new types of monoliths will also include the study of present or alterative types of polymers, in order to come out with a wider range of pore sizes, surface areas and new morphologies that can be used in this type of affinity chromatography (Pfaunmiller et al., 2013). Finally, monolithic stationary phases are expected to have a great impact on future applications, for instance if organic monolithic supports will be combined with hybrids of silica (Pfaunmiller et al., 2013).
...bioanalytical systems which based on electrochemiluminescence detection and evanescent field fluorescent detection showed the best sensitivities (Dupuy et al., 2009). The power of immunoblotting is extended with this method in order to provide a quantitative analysis of differential expression of active and parental proteins (Tibes et al., 2006). Moreover, by using RPPA, samples can be spotted at same time which is suitable for retrospective analysis of large number of specimens. This technique which can be used to analyze large number of proteins from each sample is suitable to analyze the population of cells that present in low numbers. On the other hand, this technique also has limitation which it only identifies to known proteins or targets only (Tibes et al., 2006). However, it stills a useful technique that use in the study of functional proteomics analysis.
Naturally derived cross-linking reagents are considered superior to chemical cross-linking demonstrating a low toxicity level after implantation [9]. Genipin (GIP), an aglycone of geniposide obtained from Gardenia jasminoides Ellis Plant, was first discovered by Huang et al. in 1998 [29]. GIP is found to be 10,000 times less toxic than GA. GIP-fixed tissues, usually show resistance against enzymatic degradation as compared to GA resulting in the development of biocompatible cross-linked products [30]. In a recent study, Wang et al. reported that GIP alleviated the xenogeneic host response of decellularized porcine liver scaffolds by reducing the proliferation of lymphocytes and their subsets, accompanied by a decreased release of both Th1 and Th2 cytokines. It was reported that cross-linking using GIP promotes the beneficial tissue repair through enhancing angiogenesis and cell migration
The term nanocarriers includes a wide range of different nanosized drug delivery systems. The oldest and at the same time the most clinically established nanocarriers are liposomes, spheres composed of an aqueous core surrounded by one or more concentric lipid bilayers. They are suited for the encapsulation of both hydrophillic and hydrophobic drugs, respectively in the aqueous core and whitin the lipid membrane (Hafner e.a. 2014). Liposomes increase thus the solubility of hydrophobic compounds, they enable trapping of drug molecules with a high potency, they reduce systemic side effects and toxicity and they attenuate drug clearance (Riehemann e.a. 2009)
Guanidines. The cyclic thiourea compound had greater inhibiting in the lung cancer cells compared to the bis-cyclic Guanidines. these compounds decreased the mRNA of PTHrp and hence decreased their transcription. The results showed that the decrease in the levels pf PHTrp was not due to cytotoxcicity on the cells , however due to a decrease in the promoter reporter concstruct specifically the P3 promoter .(19)
Life Science Core at UCLA, Martin, L., Chen, K., Johnson, L., Foley, R., & Murotake, R. (2005). Analysis of Protein Size and Subunit Composition Using SDS- Polyacrylamide Gel Electrophoresis. Los Angeles, CA.
Also when the problem moved specifically into the regime of the single cell becomes more intriguing and complex. It leads to further challenges in the field of sample preparation in order to make the technology suitable to be used for life sciences. The need for high sensitivity becomes more pressing as the even the most abundant metabolites in the cells are in the millimolar to micromolar range. Fur...