蛋白组学

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. ...

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. ...

Small Scale GST-Tag Purification Under Nature Conditions According to AMERSHAM-BIOSCIENCES-GST fusion protein Handbook (pdf)Aliquot of Cell Pellet after Induction The idea is to aliquot cells after i ...

His-tag protein purification using Ni-NTA magnetic beadsMaterials and reagentsExtraction buffer20 mM Tris-HCl pH 8.0 1 mM PMSF1 mg/ml lysozyme0.05% Triton X-100 ...

His-tag protein purification using Ni-NTA magnetic beadsMaterials and reagentsExtraction buffer20 mM Tris-HCl pH 8.0 1 mM PMSF1 mg/ml lysozyme0.05% Triton X-100 ...

Purification of Kar3 Motor Domain ProteinMaterials Induced cells (2 - 5 g pellet of pET/Kar3 in BL31(DE3)pLysS host cells) (See note #1)HEM buffer = 10 mM HEPES pH 7.21 mM MgCL21 mM DTT1 mM EGTAHEM + ...

Purification of Kar3 Motor Domain ProteinMaterials Induced cells (2 - 5 g pellet of pET/Kar3 in BL31(DE3)pLysS host cells) (See note #1)HEM buffer = 10 mM HEPES pH 7.21 mM MgCL21 mM DTT1 mM EGTAHEM + ...

Native purification of His-tagged Proteins from Sf9 cellsProtocol is for 150 ml of cell culture.1. Spin down cells and wash 1X in 10 ml ice cold PBS.2. Resuspend pellet in 20 ml lysis buffer: 50 mM Tr ...

Small scale His-Tag fusion protein purification under denaturative conditionsIntroduction High levels of expression of recombinant proteins in a bacterial system can lead to the formation of insoluble ...

Purification of KHC Motor Domain ProteinMaterials Induced cells (2 - 5 g pellet of pET/KHC in BL21(DE3)pLysS host cells)HEM buffer = 10 mM HEPES pH 7.21 mM DTT1 mM MgCL21 mM EGTAHEM + 25 mM NaCl HEM + ...