2.2. Protein analysesThe compositio

2.2. Protein analyses
The composition of protein samples was analyzed by SDS-PAGE under both reducing and nonreducing conditions. The gels were stained with Coomassie brilliant Blue G-250.

The concentrations of the recombinant proteins were determined using extinction coefficients calculated with the online protein analysis tool, Protparam (http://us.expasy.org/tools/protparam.html). We have used the following extinction coefficients: CRISPLD2_LCCL1: 6335 M−1 cm−1, CRISPLD2_LCCL2: 11835 M−1 cm−1, CRISPLD2_LCCL1+2: 18170 M−1 cm−1.

N-terminal sequencing was performed on an Applied Biosystems 471A protein sequencer with an on-line ABI 120A phenyltiohydantoin (PTH) analyzer.

2.3. Surface plasmon resonance analysis
Surface plasmon resonance measurements were performed on a BIACORE X (GE Healthcare, Stockholm, Sweden) instrument. Proteins to be immobilized were dissolved in 50 mM sodium acetate, pH 4.5 and 50 μl of 2.6 μM CRISPLD2_LCCL1+2, or 70 μl of 2.5 μM CRISPLD2_LCCL1, or 35 μl of 67.5 μM CRISPLD2_LCCL2 solutions were injected with a 5 μl/min flow rate on a CM5 sensor chip activated by the amine coupling method according to the instructions of the manufacturer.

Stock solutions of 2.5 mg/ml of lipopolysaccharide from E. coli 0127:B8 strain, 200 μM of diphosphoryl Lipid A from E. coli F583 strain were prepared in 20 mM HEPES buffer, pH 7.5, containing 150 mM NaCl, 5 mM EDTA and 0.005% Tween 20. The solutions were sonicated at 37 °C for 15 min.

For interaction measurements 80 μl aliquots of different concentrations of lipopolysaccharide or lipid A solutions were injected over the sensorchips with a 20 μl/min flow rate, followed by 10 min flow of buffer. Binding and washes were performed in 20 mM HEPES buffer, pH 7.5 containing 150 mM NaCl, 5 mM EDTA and 0.005% Tween 20. After each cycle the chips were regenerated by injection of 20 μl 50 mM NaOH with 60 μl/min flow rate. The injected lipopolysaccharide or lipid A solutions were always diluted and thoroughly vortexed just before injection.

Control flow cells were prepared by executing the coupling reaction in the presence of coupling buffer alone. Control flow cells were used to obtain control sensorgrams showing non-specific binding to the surface as well as refractive index changes resulting from changes in bulk properties of the solution. Control sensorgrams were subtracted from sensorgrams obtained with immobilized ligand. To correct for differences between the reaction and reference surfaces we have also subtracted the average of sensorgrams obtained with blank running buffer injections.

ผลลัพธ์ (อังกฤษ) 1:

Of 2.2. protein analyses
the composition of protein samples was analyzed by sds-page under both reducing and nonreducing conditions. the gels were stained with coomassie brilliant blue g-250.

the concentrations of the recombinant proteins were determined using extinction coefficients calculated with the online protein analysis tool, protparam (http://us.expasy.org/tools/protparam.html. ).we have used the following extinction coefficients: crispld2_lccl1: 6335 m-1 cm-1, crispld2_lccl2: 11835 m-1 cm-1, crispld2_lccl1 2: 18170 m-1 cm-1.

n-terminal sequencing was performed on an applied. biosystems 471a protein sequencer with an on-line abi 120a phenyltiohydantoin (pth) analyzer.

2.3. surface plasmon resonance analysis
5 μm crispld2_lccl2 solutions were injected with a 5 μl / min flow rate on a cm5 sensor chip activated by the amine coupling method according to the instructions of the manufacturer.

Stock solutions of 2.5 mg / ml of lipopolysaccharide from e. coli 0127: b8 strain, 200 μm of diphosphoryl lipid a from e. coli f583 strain were prepared in 20 mm hepes buffer, ph 7.5, containing 150 mm nacl,.5 mm edta and 0.005% tween 20. the solutions were sonicated at 37 ° c for 15 min.

for interaction measurements 80 μl aliquots of different concentrations of lipopolysaccharide or lipid a solutions were injected over the sensorchips with a 20 μl / min flow rate, followed by 10 min flow of buffer. . binding and washes were performed in 20 mm hepes buffer, ph 7.5 containing 150 mm nacl,.5 mm edta and 0.005% tween 20. after each cycle the chips were regenerated by injection of 20 μl 50 mm naoh with 60 μl / min flow rate. the injected lipopolysaccharide or lipid a solutions were always diluted and thoroughly vortexed just before injection.

control flow cells were prepared by executing the coupling reaction in the presence of coupling buffer alone.

ผลลัพธ์ (อังกฤษ) 3:

2.2.Protein analyses
The composition of protein samples was analyzed by SDS - PAGE under both reducing and nonreducing conditions. The gels were stained with brilliant Coomassie Blue G -250.

The concentrations of the recombinant proteins were determined using extinction coefficients calculated with the online protein analysis Tool, Protparam (http:/ /US.expasy. org/tools/protparam.html).We have used the following extinction coefficients: 2 _ LCCL CRISPLD 1:6335 M − − 1 cm 1, 2 _ LCCL CRISPLD 2:11835 M − − 1 cm 1 , CRISPLD LCCL 2 _ 1 2:18170 M − − 1 cm 1.

N-terminal sequencing was performed on an Applied Biosystems A 471 protein sequencer with an on-line 120 ABI A phenyltiohydantoin (PTH) analyzer.

2.3.Surface plasmon resonance analysis
Surface plasmon resonance measurements were performed on a BIACORE X (GE Healthcare, Stockholm, Sweden) instrument. Proteins immobilized were to be dissolved in 50 mM sodium acetate, 4.5 and 50 pHµ µL of 2.6 M CRISPLD LCCL 2 _ 1 2, or 70µ µM CRISPLD 2.5 L of 2 _ LCCL 1, or 35 µL of 67.5 µM CRISPLD 2_ LCCL 2 solutions were injected with a 5 µL/Min flow rate on a sensor chip CM 5 activated by the amine coupling method according to the instructions of the manufacturer.

Stock solutions of 2.5 mg/ml of lipopolysaccharide from E. coli 0127:B 8 strain, 200 µM of diphosphoryl Lipid A from E.coli F 583 strain were prepared in 20 mM HEPES buffer, 7.5 pH, containing 150 mM NaCl,5 mM EDTA Tween and 0.005 % 20. The solutions were sonicated for 15 min at 37 °C.

For interaction measurements 80 µL aliquots of different concentrations of lipopolysaccharide or A lipid solutions were injected with a 20 over the sensorchips µL/Min flow rate, followed by 10 min of flow buffer. Binding and washes were performed in 20 mM HEPES buffer, containing 7.5 150 pH mM NaCl,5 mM EDTA Tween and 0.005 % 20. After each cycle the chips were regenerated by injection of 20 L 50 mM NaOHµ µwith 60 L/Min flow rate. The A injected lipopolysaccharide or lipid solutions were vortexed thoroughly and always diluted just before injection.

Control flow cells were prepared by executing the coupling reaction in the presence of coupling buffer alone.Control flow cells were used to obtain control sensorgrams showing non-specific binding to the surface as well as refractive index changes resulting from changes in the bulk properties of solution. Control sensorgrams were subtracted from sensorgrams obtained with immobilized ligand.To correct for differences between the reaction and reference surfaces we have also subtracted the average of sensorgrams obtained with running buffer blank injections.