蛋白质技术

Preparation of cell lysates from yeast by enzymatic digestion of the cell wallMaterialsChemicalsLyticase USA)Brij 58Working buffer50 mM Tris-HCl pH 8.0 10% sorbitol ...

Preparation of cell lysates from yeast using a French PressEquipment and reagentsLysis buffer50 mM Tris-HCl pH 7.5 50-200 mM NaCundefined1 mM EDTA 5 mM DTT1 mM PMSF1 µg/ml leupeptin1 µg/ml pepstatin A ...

Preparation of cell lysates from yeast using a French PressEquipment and reagentsLysis buffer50 mM Tris-HCl pH 7.5 50-200 mM NaCundefined1 mM EDTA 5 mM DTT1 mM PMSF1 µg/ml leupeptin1 µg/ml pepstatin A ...

Solubilization and renaturation of proteins from inclusion bodiesMaterialsWash buffer50 mM Tris-HCl pH 7.5 50-200 mM NaCl ...

Preparation of cell lysates from yeast using glass beads vortexingEquipment and reagentsLysis buffer50 mM Tris-HCl pH 8.0 1% DMSO50-200 mM NaCundefined1 mM EDTA1 mM PMSF1 µg/ml leupeptin1 µg/ml pepstatin A ...

Solubilization and renaturation of proteins from inclusion bodiesMaterialsWash buffer50 mM Tris-HCl pH 7.5 50-200 mM NaCl ...

Large scale nuclear extract preparationHattoti's protocol adapted to cell culture:Hattori M Tugores A Veloz L Karin M Brenner D (1990) A simplified method for the preparation of transcrip-tionally act ...

Solutions:Buffer A (Hypotonic Buffer): 1L10 mM HEPES pH 7.9 10 ml 1M HEPES pH 7.91.5 mM MgCl2 1.5 ml 1M MgCl210 mM KCl 3.33 ml 3M KCl0.5 mM DTT 500 µl 1M DTT0.2 mM PMSF 1 ml 0.2 M PMSFBuffer B (S100 e ...

Preparation of Nuclear Extracts for Gel Shifts and Westerns Blots ...

Background: It is often difficult to obtain soluble and active proteins from expression in prokaryotes. Often overexpression leads to the production of inclusion bodies :insoluble aggregates of misfol ...

Protein Purification: Assays Specific Activity Initial FractionationA successful protein purification procedure can be nothing short of amazing. Whether you are starting off with a recombinant protein ...

Preparation of Brain Membrane Fractions for Western Blot AnalysisAll the procedures should be done at 4ºC using precooled reagents. For rat samples immediately remove brain from the cranium into ice c ...

Affinity chromatography (with HIS-tagged proteins)This protocol is described in less detail in Copeland et al Nature 379 162-165 1996.Affinity chromatography can be performed using a number of differe ...

GST Fusion Protein PrepItalics indicate optional steps especially useful for the analysis of untested proteins. GROWTH AND HARVESTING OF BACTERIAAdd 100 ul ampicillin to 100 ml LB. Inoculate and grow ...

Protein PurificationHD exon 1 16 38 56 and 82 CAGs fused with GST.Making constructs.Exon I of HD gene was amplified from partial cDNA constructs obtained from normal (16 CAGs) and patient (48 CAGs). T ...

Purification of GST-fusion proteinThis protocol uses Pharmacia glutathione sepharose matrix. Other companies also sells GST beads e.g. Sigma. 1) Spin down cells and resuspend in ice-cold PBS (40 ml pe ...

GST Fusion Protein Purification from Yeast5 ml overnight culture of your favorite yeast in your favorite medium. Inoculate 50 ml and grow 30o C shaking O/N until OD600 = 0.8 to 1.2. For SCD cultures ...

Purification Scheme for Ubiquitin Expression Lysate Step 1: Take the expression lysis supernatent and treat with concentrated acetic acid until the pH drops to between 4.5 and 5.0. The solution will ...

Purification Scheme for Ubiquitin Expression Lysate Step 1: Take the expression lysis supernatent and treat with concentrated acetic acid until the pH drops to between 4.5 and 5.0. The solution will ...

lution of GST-Fusion Protein from Glutathione Agarose1. Wash the GST-fusion protein glutathione-agarose complex once with 1% Triton X-100 in PBS.2. Resuspend the complex in 1% Triton X-100 in PBS.3. ...