Selecting Novel Inhibitors of Ras Raf Interaction by High Throughput Screening in SKY54 Dual Bait Yeast Two Hybrid

Dual Inhibition Microarray

The Ras Raf MEK ERK signaling pathway controls fundamental cellular processes including proliferation, differentiation, and survival, and the altered expression or action of components of this pathway is commonly observed in human cancers (22-24). Ras has suffered oncogenic mutations in nearly 30 of human cancers and mediates its action through interaction with downstream effector targets (25,26). Activated Ras binds to and recruits Raf-1 to the cell membrane, where Raf-1 is activated by a...

Robotic Genome Scale Protein Synthesizer Gen Decoder

We have recently established an automated protein synthesizer, GenDecoder (Fig. 5), based on our efficient and robust wheat germ cell-free translation system (18). As shown in the flow chart (Fig. 5A), the system begins with transcription of DNA templates generated by the split primer PCR (12) in multi-well microtiter plates, and can cover all steps up to protein production, being equipped with all the essentials for the purification of mRNA, such as pipetting, mixing, incubating, and...

Materials

Pet Sac Plasmid

E. coli strains JM109 and BL21 DE3 . 4. Restriction enzymes, T4 DNA polymerase, and T4 DNA ligase. 5. Agarose and DNA sequencing gel equipment. IPTG . 11. Purification buffer A 50 mM sodium acetate pH 5.0 , 2 mM dithiothreitol. 12. Purification buffer B 50 mM Tris-HCl pH 8.0 , 2 mM dithiothreitol. 13. SP Sepharose FF Amersham Biosciences, Piscataway, NJ . 14. HiLoad 26 60 Superdex 75 pg Amersham Biosciences . 19. Spermidine trihydrochloride. 22. SP6 RNA polymerase and RNase inhibitor Takara,...

To Probe Immunologic Sugar Moieties of SARSCoV

Glycan Array

The following example illustrates the use of carbohydrate microarray technology in an important research area. In this case, the above described eight-chamber subarray system was applied to construct a glycan array. This glycan array was then applied to identify immunogenic sugar moieties of a human cor-onavirus SARS-CoV . SARS-CoV is a newly identified human viral pathogen that caused an outbreak of severe acute respiratory syndrome SARS . Although substantial efforts have been made to study...

Construction of the Expression Plasmid for Full Length p53

Pqe 80l Bccp

The E. coli expression system used is based on the pQE-80L expression system. Sequences inserted into the multiple cloning site can be expressed as native proteins bearing an N-terminal hexahistidine tag upon IPTG induction of the T5 promoter in suitably transformed E. coli cells. 3.1.2. Amplification and Cloning of the Wild-Type p53 Gene As a Fusion to BCCP All DNA manipulations were carried out using standard recombinant DNA methods 18 to construct the expression plasmids, and are accordingly...

Synthesis

Nucleophilic Internal Substitution

Solid-Phase Combinatorial Synthesis of a Ligand Library Fig. 3 1. Epoxyactivation of agarose beads. The required amount of Sepharose CL-6B is washed with 40 mL of distilled water g of gel on a sinter funnel see Note 7 . The washed agarose is transferred to a 1-L conical flask, and 1 mL of distilled water g of gel is added. To this moist gel, 0.8 mL of 1 MNaOH mL of gel and 1 mL of epi-chlorohydrin mL of gel are added. The slurry is incubated for 10-12 h at 30 C on Fig. 2. Comparison...

Autodock3 p LIGANDproteinAutodockdpf l

LIGAND.protein.dlg amp Originally, AutoDock was devised to dock a single ligand per submitted job. However, it is possible to perform batch jobs to enable docking or screening of databases. The main issue here is to generate the correct grid maps, i.e., including halogens and other elements. Unfortunately, interaction potentials of these atoms are not always available for a list of available parameters, see The user can generate his or her own template GPF including the desired parameters. To...

Chemistry for Chemical Genomics

New methods and strategies have been developed to design and use small molecules that allow the functional dissection of molecular pathways, cells, and organisms by selective small-molecule ligands or modulators. In this overview, we are focusing on diversity aspects, design methods, and chemical synthesis strategies for the application of small molecules as tools for chemical genomics. Examples for different successful chemical-genomics strategies include the selection of diverse drug-like...

Determination of Protein Yield and Purity

Eluted material was assessed on an Agilent 2100 Bioanalyzer using a Protein 200 Plus LabChip Caliper to determine protein purity, concentration, and yield. However, traditional SDS-PAGE can also be used, if necessary. The LabChip system is a fluorescence-based detection system that offers the advantages of requiring only 1-4 pL of protein samples, as compared to 10 pL required for equivalent band identification on traditional SDS-PAGE gels, and results for a set of 10 samples are obtained in 30...

Split Primer Design for the Construction of PCRGenerated Linear Template DNAs for Transcription and Translation

Cloning of cDNA into an expression vector is one of the most laborious and time-consuming steps of recombinant protein synthesis. Attempts have been made to direct synthesis from PCR-generated linear cDNAs 18,36 . These methods generally involve the incorporation of transcriptional and translational start and stop signals into primers for the generation of autonomous coding fragments by PCR. In order to establish a general and practical method for the cellfree expression of proteins, we focused...

Options FlexX Browse results

A window is opened in which the jobname used earlier to submit the run can be selected and the ligands with their corresponding FlexX score will be displayed. The Show Failed Ligand button gives ligands for which no docking solution has been obtained. The successfully docked ligands can be sorted according to their binding energy score by clicking the Sorted icon. 3.4.2. Analyzing the Results Generated by Gold The docking poses found by GOLD are stored in MOL2 format. The ligand conformations...