I thought some practical tips on protein
labeling might interest people. Usually
with Thiol labeling we're aiming to get a
probe in just one place on a protein. So,
you may be lucky and have a protein that
has a naturally reactive cysteine like
like actin for example, which has one
reactive near the C-terminus. It's
getting more common now for people who
really want to study a protein in detail
for biophysics to actually engineer out
all the surface exposed cysteines and
then engineer in one place a reactive
cysteine. So that's some amount of work
on the cloning that can be worth
it and you can actually walk a
fluorochrome in two different places on
the molecule by depending on where you
put the cysteine. Now it's really
important to protect your cysteine until
you do the labeling. So you want to
include reducing agent in all the
buffers until the labeling step and then
remove your DTT or β-mercaptoethanol
in the last step or else titrate
it out with probe. Cysteine labeling is
typically done at pH 7 to 8. If you go
too high in a pH you risk labeling
tyrosine residues. The nice
thing about these cysteine probes
is they are
not very reactive with water so you
don't need to use very much. Just enough
essentially, 1.5 to 3 equivalents is
typical. And because you're not using
very much you have to react for a long
time overnight or room temperature. So
these are kind of cookbook recipes. With
the typical lysine labeling reaction
where you're not usually aiming for
site-specific, it's the
considerations are a little bit different.
There's plenty of surface lysine on
most proteins so you can often want to
label several of them. For example on an
antibody you might want to aim for about
4 fluorescent molecules per antibody.
Usually the antibody will still be fine
and you get plenty of fluorescence then.
These NHS ester probes, unlike the Thiol
probes, they're quite chemically reactive.
It's important to store them in a
high-quality dry solvent. DMSO is the
most common, or dry in the freezer. We
often aliquot them in DMSO and store
them at minus 20. You want to label at
neutral pH or above.
You want to use a non-nucleophilic
buffer. We often use HEPES in my lab,
bicarbonate, borate. Important not to use
DTT or thiol. I mentioned they'll
decompose the probe. The issue of the pH
is this: the higher your pH, the
faster you'll react with the protein but
also, the faster the probe will be
decomposed by hydroxide ions. And in our
experience it often actually works a
little better at let's say pH 8 than it
does at pH 10 because you start getting
to rapid hydrolysis. And just as a rule
of thumb here, these NHS ester probes,
their half-life in solution will be a
minute or two at pH 9 and several
hours at pH 7, well about an hour or
something at pH 7. So, if you do a pH 9
reaction it's all over in an hour or
something, if you do a pH 7 reaction you
may want to leave it overnight, say at 4°.
Now, if you want to control the
stoichiometry, you don't want too much
label. If you overlabel a protein it may
quench the solution or be
non-fluorescent. With the Thiols, you
usually do this by stoichiometry, so you
add 1.5 moles per protein, or not too
much anyway. With the Amine reactive
probes, because they're hydrolyzing,
you're often controlling the reaction
not by stoichiometry, not by so much
so many moles per mole of protein but
rather by concentration. But in practice,
whether it's more stoichiometry limit, or
more concentration limited, depends on
the concentration of your protein and so
you may have to pilot it. The good news
about reacting tough proteins like
antibodies is it turns out they'll
accommodate a fairly wide range of
protein density so it's actually fairly
easy to get a good labeled antibody.
A few other practical points. Your protein
may have some essential cysteines or
lysines. I have spent half my life
working on the molecule tubulin and it
turns out if you take tubulin dimer form
in solution, it has highly reactive
cysteines and lysines that are essential
for its polymerization function and
there are some tricks for protecting
those. So, you can sometimes sterically
blockade essential cysteines or lysines
by having a high concentration of a
physiological ligand that blocks
access of the labeling reagent. So, for
example in the case of tubulin, you have
to label in the polymer. So, if the tubulins
polymerize into a microtubule
that actually protects these essential
groups. And sometimes with an enzyme you
can protect by having a high amount of
substrate that protects the active site.
I think my final practical point is
separating free probes from protein.
Traditionally you do this by molecular
size by dialysis, which is kind of slow,
or by gel permeation chromatography. With
these modern dies with the sulfonate
groups that works quite well. But if you
have a more sticky die it often will
stick to the protein and be hard to
separate. And it's useful for check how
much is left by chromatography on silica
gel. Increasingly, in my lab we like to use
an affinity based method labeling either
in solution, or first attaching the
protein and labeling it on a column. So
for example, with antibody labeling we're
often attaching the protein, to a protein,
an antibody to a protein A column,
labeling it on column, flushing away the
excess and then eluting the antibody.
That seems to work really well and give
us very little free die.