SEC
16
Applications of TSKgel SW-type Gel Filtration columns
Membrane Proteins
A TSKgel G3000SW column was used to study the effect of different
concentrations of the non-ionic surfactant octaethyleneglycol
dodecylether on the analysis of membrane proteins from a crude extract
from rat liver microsome. The effect of different concentrations of
surfactant on the separation of membrane proteins is seen in
FIGURE 8
.
As the concentration of octaethyleneglycol dodecylether increases
to 0.05%, the main peak becomes sharper and recovery increases.
Caution: we recommend that columns that have been used with a
surfactant-containing mobile phase are dedicated for that particular
use.
NUCLEIC ACIDS
Separation of four E. coli RNAs, shown in
FIGURE 9
, confirms the high
performance of TSKgel G4000SW columns for samples with a wide high
molar mass range. The sample consists of 4S tRNA (2.5 × 10
4
Da), 5S
rRNA (3.9 ×10
4
Da), 16S rRNA (5.6 × 10
5
Da), and 23S rRNA (1.1 × 10
6
Da).
All four polynucleotides are within the molar mass range recommended
for this TSKgel SW column.
Separation of membrane protein by SEC with different
surfactant concentration in the eluent
Minutes
Column:
Sample:
Elution:
Flow Rate:
Detection:
TSKgel G3000SW, 7.5mm ID x 60cm
Membrane protein from a crude extract from
rat liver microsome
(0.2mol/L sodium chloride + 20% glycerol +
octaethyleneglycol dodecylether) in 50mmol/L
phosphate buffer,pH7.0. Note: concentration
of surfactant: (1) 0.005%, (2) 0.01%, (3) 0.025%,
(4) 0.05%
1.0mL/min
UV @ 280nm
10
20
30
40
(1)
(2)
(3)
(4)
figure 8
Analysis of membrane protein with differing surfactant
concentrations in the mobile phase
Column: TSKgel G3000SW, 10 µm, 7.5 mm ID × 60 cm
Mobile phase: (0.2 mol/L sodium chl ride + 20% glycerol + octaethylene
glycol dodecylether) in 50 mmol/L phosphate buffer, pH 7.0;
Note: concentration of surfactant: 1.) 0.005% 2.) 0.01% 3.) 0.025% 4.) 0.05%
Flow rate: 1.0 mL/min; Detection: UV @ 280 nm
Sample: membrane protein from a crude extract from rat liver microsome
1
2
4
3
20
30
40
50
Retention time (minutes)
Detector response (AU)
figure 9
Separation of total E. coli RNA
Columns: TSKgel G4000SW, 13 µm, 7.5 mm ID × 30 cm × 2
Mobile phase: 0.13 mol/L NaCl in 0.1 mol/L phosphate buffer,
pH 7.0, + 1 mmol/L EDTA
Flow rate: 1.0 mL/min; Detection: UV @ 260 nm; Injection vol.: 5 µg
Sample: 0.1 mL of 1:10 diluted solution of total E. coli RNA:
1. 23s rRNA (1.1 × 106 Da); 2. 16s rRNA (5.6 × 105 Da)
3. 5s rRNA (3.9 × 104 Da); 4. 4s rRNA (2.5 × 104 Da)
