化学生物学实验技术

Typical modification sites: RGG box or RXR sequence motifs R-arginine G-glycineX-any aminoacid.Enzymes catalysing protein arginine methylation: PRMT1 from rat and the humanhomologue HRMT1L2 human PRMT ...

Purpose and backgroundsPrinciple of radioiodinationAddition of oxidizing reagents (such as chloramine-T or peroxidase+H202) converts I- to I+ or I3-. This highly reactive molecule attacks o-position o ...

Motor proteins of several kinesin family groups have now been crystallized: monomeric Kinesin-1 motor domains from human rat and Neurospora (Kull et al. 1996; Sack et al. 1997; Song et al. 2001) and d ...

Materials Milipore filter type HA 0.45 micronCulture plates (Linbro model 76-033-05)Protein in DDW or HEPES buffer (10 mg/mL)Vacuum grease Syringe with 18 G needleProcedure 1. Millipore filter all sol ...

Background:Proteins like many molecules can be prompted to form crystals when placed in the appropriate conditions. In order to crystallize a protein the purified protein undergoes slow precipitation ...

This specfic protocol is the latest incarnation of peptide mapping procedures that have been developed here in the TVL/MBVL of the Salk Institute over the last 20 years. Wade Gibson developed the firs ...

In identifying peptides from proteins with a known sequence it is often useful to be able to predict how a peptide will migrate during electrophoresis chromatography or two-dimensional peptide mapping ...

1. Immunoprecipitate the protein and run it on a preparative gel. CNBr cleavage must be done with protein transferred to a nitrocellulose filter. Neither Immobilon nor Nylon can substitute. IMPORTANT: ...

Materials: Overlay/Rehydration buffer:125 mM Tris-HCl pH 6.8 1mM EDTA 0.1% SDS 1mM 2-b-mercaptoethanol30% glycerolsome Bromophenol blueFor 1 ml you will need0.35 ml of 2X buffer0.30 ml H20 0.30 ml gly ...

1) Grow cells to a density of 5-8 X 105 cells/ml in RPMI 1640 containing serum.2) Pellet cells and wash 1 time with room temperature PBS.3) Resuspend final pellet in an appropriate volume of prewarmed ...

Buffers:- 2X buffer10 ml 0.5 M imidazol pH 6.60.21 g LiCl1.25 ml 1 M MgCl21.0 ml 0.1 M EGTA pH 6.6-- Bring volume up to 50 ml with distilled water.- dilution buffer1 ml 0.5 M imidazol pH 6.62.5 ml 20 ...

1) Remove 2 500 µl aliquots of supernatant into scintillation vials add scintillation fluid and count.2) Aliquot 1.6 mls of the remaining supernatant into each of two pyrex glass tubes.3) Add 6 mls of ...

Packing column:1) To 20 g of 100-200 mesh Bio-Sil A silica gel add 80 mls of chloroform.2) Place a small portion of glass wool at the base of the column.3) Pour gel solution into column and use a stir ...

1) Prepare a C-18 reverse phase prep-sep cartridge by washing with:a) 10 ml of methanolb) 20 ml of chloroform-methanol (2:1)c) 10 ml of methanold) 10 ml of methanol-1.6 M sodium acetate (1:1)2) Apply ...

Reagents:Bacterial strainE. coli N4830/pJW10LB amp media50 µg/ml ampicillinHigh salt bufferfor 1 L50 mM KH2PO4 6.8 g150 mM KCl 11.18 g50 mM sodium pyrophosphate (Na4P2O7-10H2O) 22.3 g5 mM Na2 EDTA 1.8 ...

1) Incubate cells with 1 µCi/ml of 3H-galactose for 72 hours.--- If treatment is for an extended period of time: treat in serum free media containing labeled galactose.2)Wash label out and spin cells ...

Reagents:Lysis buffer25 mM Tris-HCl pH 7.45 mM EDTA1 mM ATP20 µg/ml CLAP1 mM PMSFBuffer A10 mM MgCl20.2 M Tris-HCl pH 7.40.2 % Triton X-100Buffer B0.2 M sodium acetate pH 5.00.2 % Triton X-100Buffer C ...

As insects have an open circulatory system the hemolymph can be simply collected through an incision in the body wall. Most conveniently you should cut a leg and let the blood drip into a chilled glas ...

Synthesis reaction:1) Mix purified DMSM with cyclohexylamine and 14CH3I at a ratio of 1/1.1/1.3 in 5 ml of methanol.2) Allow reaction to proceed at room temperature for atleast 18 hours in the dark.3) ...

NOTE - Use arachidonic acid from Biomol (# FA-003)1) Make up the appropriate concentration of AA in 100% ethanol.2) Add 1µl of phenol red solution.-- The solution should appear yellow since the free a ...