Biology Reference
In-Depth Information
23. Coomassie Brilliant Blue G is a sensitive stain for protein
detection in PAGE gels. Coomassie staining gives blue bands
on a background, with a sensitivity of 50-100 ng/band. All
steps are done on a shaker with gentle mixing.
24. The optimal quantity of protein to load varies widely depending
on factors such as sample complexity, the length (7, 11, 13, 18,
and 24 cm) and pH range (from very acidic proteins at pH 3
to extremely basic proteins at pH 11) of the Immobiline
DryStrip gel, and the method of visualizing the two-dimen-
sional gel separations. The volume of rehydration solution
used to solubilize the sample depends on the sample loading
method (sample in-gel rehydration or cup-loading) and the
length of the Immobiline DryStrip gel used for the fi rst-dimen-
sion separation.
25. For increased resolution between pH 5 and pH 7, use a non-
linear gradient pH 3-10 strip (pH 3-10 NL) to distribute the
proteins more evenly over the strip.
26. Remove the protective cover from the surface of the IPG strip
starting at the acidic (+) to prevent damage to the basic end of
the gel, which is generally softer.
27. The pointed end of the holder is over the anode (+) (pointed
to the back of the unit) and the blunt end over the cathode (−).
Guide marks along the sides of the platform show approximate
positioning for each strip holder size (7, 11, 13, 18, and
24 cm). Check that each of the two external electrode contacts
makes metal-to-metal contact.
28. Overlay the resolving gel with water for gels having acrylamide
concentration lower than 8 % and use isobutanol or isopropa-
nol saturated with water for gels of 10 % or greater. This overlay
prevents contact with atmospheric oxygen (which inhibits
acrylamide polymerization) in addition to helping to level the
resolving gel solution.
29. If necessary, store either tryptic peptides before or after desalting
step at −20 °C (<24 h) or −80 °C (for long periods).
30. Prepare a fresh matrix solution each time or a stored solution
at room temperature no longer than 48 h. Perform always a
spin centrifugation to remove the non-dissolved matrix.
31. An additional control is put onto the MALDI target consisting
of a trypsin digest from BSA protein. Calibration is performed
according to a quadratic regression curve taking into account
four or fi ve points that bracketed the mass range of interest.
32. It is recommended to examine different regions of the spot to
fi nd those giving superior signal-to-noise ratios.
33. Database searching can be speeded up by using a restricted
database. This database can be created with a subset of National
