Optimizing blotting
Optimizing blotting
When using the iBlot
™
2 Gel Transfer Device, most proteins transfer efficiently using
the protocol in this manual. Based on specific properties of a protein or a set of
proteins, some optimization of the blotting protocol may be necessary.
Optimize blotting as follows:
Perform an ethanol equilibration step prior to transfer
To improve the transfer of high-molecular weight proteins from mini- or midi-
NuPAGE
™
or Tris-Glycine gels, submerge the gel in 20% ethanol (prepared in
deionized water), and equilibrate for 5–10 minutes at room temperature on a shaker
prior to transfer.
Do not equilibrate for longer than 10 minutes, or sensitivity may be reduced. After
equilibration, perform transfer using the iBlot
™
2 Gel Transfer Device as described in
this manual.
Increase or decrease transfer time
Based on the initial results, you can increase or decrease the transfer time for the
Method used to perform the transfer (refer to Chapter 3, “Custom methods“ for
details on customizing a Method).
Do not perform transfer for more than the recommended run time limit indicated for
each Method (“Description of methods“ on page 23).
Proteins >150 kDa migrate more slowly, and require more time to transfer. If your
protein of interest is in this size range, it may be necessary to use a Run Time of 8–
10 minutes for your transfer.
Small proteins <30 kDa migrate more rapidly during electrophoretic separation, and
consequently require less time to transfer from the gel matrix to the membrane. If
your protein of interest is in this size range, you may need to reduce the Run Time to
5–6 minutes for your transfer.
It is normal for some proteins to remain in the gel, because some high molecular
weight proteins do not transfer completely using the iBlot
™
2 Gel Transfer Device as
compared to wet transfer apparatus.
Since the sensitivity of detection using the iBlot
™
2 Gel Transfer Device is higher than
that of semi-wet and semi-dry blotting, complete transfer of proteins is not required.
Near-complete transfer of prestained standard protein bands is observed with the
iBlot
™
2 Gel Transfer Device. However, note that the complete transfer of prestained
protein standards does not indicate complete transfer of other proteins or blow-
through of other proteins.
C
iBlot
™
2 Dry Blotting System User Guide
49
