Protein Protocols Protein Protocols

DIGE 195
4. The presence of Pharmalytes in the lysis buffer interferes with labeling as some contain
amines which react with and inactivate the dye. Similarly, the presence of any other
primary amine containing compound (such as Tris) should be avoided.
5. As in Note 2, there’s a danger of cyanate formation in this buffer. It is thus preferable to
add the acetic acid at the same time as the urea. The lower pH will slow down the
breakdown process.
6. This buffer may be reused up to three times or until the DTT is no longer detectable by its
smell, whichever comes first.
7. We typically do not use protease inhibitors. The combination of the lysis buffer with its
reducing ability, the chaotropic effects of the urea and the surfactant, and the cold temperature
seems to inactivate proteolytic activity. We also do not perform any steps requiring
room temperature or protein activity (such as the DNAse/RNAse treatment found in
some protocols). Furthermore, the presence of the inhibitors may sometimes interfere with
the fluorescent labeling.
8. The sample cups (see Subheading 3.2.) on the IEF gel have about 100 µL maximum
capacity. However, if necessary, more volume can be handled by ordering more sample
cup holder bars separately from the Dry strip kit and spreading one sample between several
cups. As IEF is a focusing technique, sample does not necessarily be all applied in
exactly the same spot.
9. The dye synthesis is detailed elsewhere (6). The dyes are not commercially available as of
the time of writing.
10. Try not to get any water on the gel side as this makes the gel very sticky, which causes
problems when the strip is being rolled onto the glass plate. If the gel side does get wet,
sometimes the situation can be saved by dabbing the water gently with a paper towel.
Take care to not touch the paper to the gel. If you know you have sticky gels, then hold
down the strips at the basic end while rolling.
11. Rehydration should continue for a minimum of 8 h. The gels should not be used after 24 h
in the rehydration solution.
12. Getting the wicks to the exact size is not that important—eyeball accuracy is good enough.
Also, even though Pharmacia supplies and sells “special” wick paper, we have found that
3MM Whatman chromatography paper will work just as well.
13. This is probably one of the more tricky aspects of the IEF procedure. It is important not to
get the wicks too dry or to leave them too wet. Sparking is usually caused by the wicks
being at the wrong dampness. Following these steps usually works, but work pace can
play a role, as can the ambient humidity. One way of checking to see if the wicks are
“correct” is to remove one from the damp patch and touch it lightly to a dry area on the
paper. If it leaves behind a slight imprint, then it is most likely at the right dampness. If too
wet, place firmly and dry a little more before placing on the gel. If too dry, rehydrate the
wicks and repeat, waiting for half the time as the first try.
14. The Teflon membranes replace the paraffin oil which is normally used to cover the gels to
isolate and keep the gels dry. We have found that using the oil produces a host of problems
of its own. The membranes work just as well and are easy to use and remove without the
messiness of the oil.
15. It is preferable to use a swingout rotor to get a more compact pellet and minimize loss of
yeast cells. We use a benchtop centrifuge with a swingout “TechnoSpin” rotor from Sorvall
Instruments.
16. Do not press on the sample cups too hard. The gel is quite delicate and making a dent or
hole in it is as deleterious as not having the sample cup seated properly. Even if the sample
does leak, this is not as big a tragedy as it might seem at first. As long as the sample stays