Bio-Rad ReadyPrep™ 2-D Starter Kit - Manual (Page 16)

6. Using Table 3, add the indicated volume of complete equilibration buffer II (containing
iodoacetamide) to each strip.
7. Return the tray to the orbital shaker for 10 minutes.
8. During the incubation, melt the overlay agarose solution in a microwave oven using the
following method.
a Loosen the cap of the bottle of overlay agarose and place the bottle in the center of a
microwave oven.
b Microwave on high 45 – 60 seconds until the agarose liquifies. Times will vary depend-
ing upon the power of the microwave. It is best to stop the microwave after 30 seconds
and swirl the bottle to mix the solution. Then, return the bottle and microwave for 15
additional seconds, swirl the liquid again and repeat this again if needed. CAUTION
should be taken as the overlay agarose contains SDS, which when heated can cause
bubbling over of the agarose solution. Set the overlay agarose solution aside. If
desired a stirbar can be added and the bottle set to stir slowly.
9. Discard the equilibration buffer II by decanting at the end of the incubation period as
described in step 5 above.
4.8 SDS-PAGE.
1 Fill a 100 ml graduated cylinder or a tube that is the same length or longer than the IPG
strip length with 1X Tris-glycine-SDS running buffer. Use a Pasteur pipette to remove any
bubbles on the surface of the buffer.
2 Finish preparing the SDS-PAGE gels by blotting away any excess water remaining inside
the IPG well using Whatman 3MM or similar blotting paper. Lay the gels onto the bench
with the top of the gel facing you and the back (tall) plate on the bottom; see Figure 10.
14
Strip Length 7 cm 11 cm 17 cm
Equilibration Buffer I 2.5 ml 4 ml 6 ml
Equilibration Buffer II 2.5 ml 4 ml 6 ml
Table 3.
Fig. 10. Rinsing the ReadyStrip in 1 X TGS running buffer.