2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol

反応条件: temp:95.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Samples were diluted to 1 mg/ml with the relevant buffer and 16.71a1 added to 12.5 μl of 4×LDS sample buffer (Invitrogen), 15.8 μl of Milli-Q water and 5 μl reducing agent (Invitrogen). The samples were heated at 95° C. for one minute and then placed on ice. 15 μl of each sample was loaded onto a 4-12% BisTris gel (Invitrogen) in a tank containing 1×MES SDS running buffer and the gel run for 35 minutes at a constant current of 500 mA. After electrophoresis the gel was removed from its casing, rinsed for 3×10 minutes with Milli-Q water, stained with Gelcode® Blue staining reagent (Pierce) for a minimum of one hour and then destained with Milli-Q water. The gel was photographed and analysed using a UVP GDS8000 gel documentation system. The relative abundance of BAK502G9 heavy and light chain in each sample was determined. Samples were diluted to 1 mg/ml with the relevant buffer and 16.71a1 was removed from its casing rinsed for 3×10 minutes with Milli-Q water stained with Gelcode® Blue staining reagent (Pierce) for a minimum of one hour

反応条件: temp:95.01; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Samples were diluted to 1 mg/ml with the relevant buffer and 16.71a1 added to 12.5 μl of 4×LDS sample buffer (Invitrogen), 15.8 μl of Milli-Q water and 5 μl reducing agent (Invitrogen). The samples were heated at 95° C. for one minute and then placed on ice. 15 μl of each sample was loaded onto a 4-12% BisTris gel (Invitrogen) in a tank containing 1×MES SDS running buffer and the gel run for 35 minutes at a constant current of 500 mA. After electrophoresis the gel was removed from its casing, rinsed for 3×10 minutes with Milli-Q water, stained with Gelcode® Blue staining reagent (Pierce) for a minimum of one hour and then destained with Milli-Q water. The gel was photographed and analysed using a UVP GDS8000 gel documentation system. The relative abundance of BAK502G9 heavy and light chain in each sample was determined. Samples were diluted to 1 mg/ml with the relevant buffer and 16.71a1 was removed from its casing rinsed for 3×10 minutes with Milli-Q water stained with Gelcode® Blue staining reagent (Pierce) for a minimum of one hour

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Protein samples were prepared for SDS-PAGE by diluting 1:1 with 2× denaturing buffer (40 μg/ml SDS, 20% glycerol, 30 μg/ml DTT and 10 μg/ml bromophenol blue in 125 mM Tris, pH 6.8) and boiling for 2 min. Pre-cast Nu-PAGE 10% Bis-Tris protein gels were obtained from Invitrogen. Each lane was loaded with a maximum of 10 μl of sample and run for 50 min at 200V. After electrophoresis was complete, the gel was stained with Invitrogen SafeStain (1 hour in staining solution, then washed overnight with water).

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Protein samples were prepared for SDS-PAGE by diluting 1:1 with 2× denaturing buffer (40 μg/ml SDS, 20% glycerol, 30 μg/ml DTT and 10 μg/ml bromophenol blue in 125 mM Tris, pH 6.8) and boiling for 2 min. Pre-cast Nu-PAGE 10% Bis-Tris protein gels were obtained from Invitrogen (Carlsbad, Calif.). Each lane was loaded with a maximum of 10 μl of sample, and run for 50 min at 200V. After electrophoresis was complete, the gel was stained with Invitrogen SafeStain (1 hour in staining solution, then washed overnight with water).

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Five micrograms of each sample were loaded on 12% NuPAGE® Bis-Tris gels, and electrophoresed under reducing conditions at 200 V in NuPAGE® MOPS SDS Running buffer.

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Protein samples were prepared for SDS-PAGE by diluting 1:1 with 2× denaturing buffer (40 μg/ml SDS, 20% glycerol, 30 μg/ml DTT and 10 μg/ml bromophenol blue in 125 mM Tris, pH 6.8) and boiling for 2 min. Pre-cast Nu-PAGE 10% Bis-Tris protein gels were obtained from Invitrogen. Each lane was loaded with a maximum of 10 μl of sample and run for 50 min at 200V. After electrophoresis was complete, the gel was stained with Invitrogen SafeStain (1 hour in staining solution, then washed overnight with water).

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Protein samples were prepared for SDS-PAGE by diluting 1:1 with 2× denaturing buffer (40 μg/ml SDS, 20% glycerol, 30 μg/ml DTT and 10 μg/ml bromophenol blue in 125 mM Tris, pH 6.8) and boiling for 2 min. Pre-cast Nu-PAGE 10% Bis-Tris protein gels were obtained from Invitrogen (Carlsbad, Calif.). Each lane was loaded with a maximum of 10 μl of sample, and run for 50 min at 200V. After electrophoresis was complete, the gel was stained with Invitrogen SafeStain (1 hour in staining solution, then washed overnight with water).

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Five micrograms of each sample were loaded on 12% NuPAGE® Bis-Tris gels, and electrophoresed under reducing conditions at 200 V in NuPAGE® MOPS SDS Running buffer.

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Reactions were assembled in a volume of 260 μl containing 50 mM Hepes buffer, 150 mM NaCl, pH 7.5 and as indicated 5 μM SrtADN in 50 mM BisTris, pH 7.5, 10 μM LPXTG peptide (DABCYL-QALPETGEE-EDANS), 10 μM TGXLP peptide (DABCYL-QATGELPEE-EDANS), 5 mM MTSET, 0.2 M NH2OH, 5 mM pHMB or 10 mM DTT. Incubations were carried out for 1 hour at 37° C. Samples were analyzed in a fluorimeter using 395 nm for excitation and 495 nm for recordings.

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Reactions were assembled in a volume of 260 μl containing 50 mM Hepes buffer, 150 mM NaCl, pH 7.5 and as indicated 5 μM SrtADN in 50 mM BisTris, pH 7.5, 10 μM LPXTG peptide (DABCYL-QALPETGEE-EDANS), 10 μM TGXLP peptide (DABCYL-QATGELPEE-EDANS), 5 mM MTSET, 0.2 M NH2OH, 5 mM pHMB or 10 mM DTT. Incubations were carried out for 1 hour at 37° C. Samples were analyzed in a fluorimeter using 395 nm for excitation and 495 nm for recordings.

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Protein samples were prepared for SDS-PAGE by diluting 1:1 with 2× denaturing buffer (40 μg/ml SDS, 20% glycerol, 30 μg/ml DTT and 10 μg/ml bromophenol blue in 125 mM Tris, pH 6.8) and boiling for 2 min. Pre-cast Nu-PAGE 10% Bis-Tris protein gels were obtained from Invitrogen (Carlsbad, Calif.). Each lane was loaded with a maximum of 10 μl of sample and run for 50 min at 200V. After electrophoresis was complete, the gel was stained with Invitrogen SafeStain (1 hour in staining solution, then washed overnight with water).

反応条件: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

実験手順: Protein samples were prepared for SDS-PAGE by diluting 1:1 with 2× denaturing buffer (40 μg/ml SDS, 20% glycerol, 30 μg/ml DTT and 10 μg/ml bromophenol blue in 125 mM Tris, pH 6.8), and boiling for 2 min. Pre-cast Nu-PAGE 10% Bis-Tris protein gels were obtained from Invitrogen (Carlsbad, Calif.). Each lane was loaded with a maximum of 10 μl of sample, and run for 50 min at 200V. After electrophoresis was complete, the gel was stained with Invitrogen SafeStain (1 hour in staining solution, then washed overnight with water).