Combination of 2DE and LC for Plant Proteomics Analysis - Plant Proteomics: Methods and Protocols - page 127

pH 3-10 NL IPG strips

Wheat

3 4 5 6 7 8 9 10

homogenized

pH

Embryos Proteins

IPGphor
Isoelectrofocusimg

SDS-PAGE/ 12% PAA gels

Strip
Rehydratacion
and IEF

18 cm X 20 cm staining

Image Master TM 2D
Platinum Software.

p I

3 10

kDa

66

45
36
29
24

MW

20

14

Fig. 1 Summary of extraction and separation in 2DE. First dimension with IPG linear pH 3-10 and second
dimension on 12 % SDS-PAGE. Total proteins stained with CBB R250. After gel image analysis using the
ImageMaster Platinum software, 768 spots were detected in the pH 4-8 gradient vs. the 899 detected in the
pH range 3-10. The relative molecular mass standards (×1,000 Da) are indicated in the Y -axis and the pI is
given at the top. Gels are of 24-cm length

Flow rate, 0.2 ml/min. Temperature, 25-30 °C. Detection,
UV 280 nm and back pressure, 600-1,000 psi.
3. After equilibration, the sample was introduced with a manual
injector onto the column. In the fi rst dimension the proteins
were bound to a strong anion exchanger and eluted with a
continuous decreasing pH from 8.0 to 4.0 (elution buffer).
The pH began to decrease after 40-45 min.