9
Prepare the two equilibration solutions from the SERVA IPG Strip equilibration
buffer:
DTT solution: Weigh urea and Dithiothreitol (DTT), add the equilibration buffer
and dissolve completely.
IAA solution: Weigh urea and Iodoacetamide (IAA), add the equilibration buffer
and dissolve completely.
Sufficient amount for 1 x 18cm, 1 x 24 cm, 2 x 11cm, 3 x 7cm
Urea [g] DTT [mg] IAA [mg] Eq. Buffer [ml] Total [ml]
IAA solution 1.8 - 125 5 6
Equilibrate each strip in 6 ml (18 or 24 cm strips), 3 ml (11 cm strips) or 2 ml (7
cm strips) solution in an equilibrator on an orbital shaker with 30 –50 /min in DTT
for 15 min and in IAA solution for 15 min.
2) Applying the IPG-strip
IPG strips should still contain equilibration buffer on their surfaces. Do not blot
IPG strips dry, this can cause vertical streaking in the second dimension due to
insufficient protein transfer. Some IPG strips have rather long protruding support
film on the ends: In this case the plastic film support on both sides of the IPG-strip
must be trimmed just beyond the gel.
The strip should be carried horizontally and applied to the slot center first.
The strip should be placed in the IPG slot, gel side down, with the anodal side to
the right. To ensure good contact in the slot the back of the forceps is slid gently
along the back of the IPG strip.
DOMINIQUE DUTSCHER SAS
To Next Page
Loading...