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Labeling Proteins with Fluorescent Probes

00:00:10.00 I thought some practical tips on protein
00:00:13.00 labeling might interest people. Usually
00:00:16.00 with Thiol labeling we're aiming to get a
00:00:18.00 probe in just one place on a protein. So,
00:00:21.00 you may be lucky and have a protein that
00:00:23.00 has a naturally reactive cysteine like
00:00:25.00 like actin for example, which has one
00:00:27.00 reactive near the C-terminus. It's
00:00:31.00 getting more common now for people who
00:00:33.00 really want to study a protein in detail
00:00:35.00 for biophysics to actually engineer out
00:00:37.00 all the surface exposed cysteines and
00:00:39.00 then engineer in one place a reactive
00:00:42.00 cysteine. So that's some amount of work
00:00:43.00 on the cloning that can be worth
00:00:46.00 it and you can actually walk a
00:00:47.00 fluorochrome in two different places on
00:00:49.00 the molecule by depending on where you
00:00:51.00 put the cysteine. Now it's really
00:00:53.00 important to protect your cysteine until
00:00:55.00 you do the labeling. So you want to
00:00:56.00 include reducing agent in all the
00:00:58.00 buffers until the labeling step and then
00:01:00.00 remove your DTT or β-mercaptoethanol
00:01:02.00 in the last step or else titrate
00:01:05.00 it out with probe. Cysteine labeling is
00:01:07.00 typically done at pH 7 to 8. If you go
00:01:10.00 too high in a pH you risk labeling
00:01:13.00 tyrosine residues. The nice
00:01:16.00 thing about these cysteine probes
00:01:18.00 is they are
00:01:19.00 not very reactive with water so you
00:01:21.00 don't need to use very much. Just enough
00:01:23.00 essentially, 1.5 to 3 equivalents is
00:01:26.00 typical. And because you're not using
00:01:27.00 very much you have to react for a long
00:01:30.00 time overnight or room temperature. So
00:01:32.00 these are kind of cookbook recipes. With
00:01:36.00 the typical lysine labeling reaction
00:01:38.00 where you're not usually aiming for
00:01:40.00 site-specific, it's the
00:01:44.00 considerations are a little bit different.
00:01:45.00 There's plenty of surface lysine on
00:01:48.00 most proteins so you can often want to
00:01:50.00 label several of them. For example on an
00:01:52.00 antibody you might want to aim for about
00:01:54.00 4 fluorescent molecules per antibody.
00:01:57.00 Usually the antibody will still be fine
00:01:59.00 and you get plenty of fluorescence then.
00:02:01.00 These NHS ester probes, unlike the Thiol
00:02:04.00 probes, they're quite chemically reactive.
00:02:06.00 It's important to store them in a
00:02:08.00 high-quality dry solvent. DMSO is the
00:02:11.00 most common, or dry in the freezer. We
00:02:15.00 often aliquot them in DMSO and store
00:02:17.00 them at minus 20. You want to label at
00:02:21.00 neutral pH or above.
00:02:23.00 You want to use a non-nucleophilic
00:02:26.00 buffer. We often use HEPES in my lab,
00:02:29.00 bicarbonate, borate. Important not to use
00:02:31.00 DTT or thiol. I mentioned they'll
00:02:33.00 decompose the probe. The issue of the pH
00:02:36.00 is this: the higher your pH, the
00:02:39.00 faster you'll react with the protein but
00:02:40.00 also, the faster the probe will be
00:02:43.00 decomposed by hydroxide ions. And in our
00:02:47.00 experience it often actually works a
00:02:48.00 little better at let's say pH 8 than it
00:02:50.00 does at pH 10 because you start getting
00:02:53.00 to rapid hydrolysis. And just as a rule
00:02:55.00 of thumb here, these NHS ester probes,
00:02:58.00 their half-life in solution will be a
00:03:00.00 minute or two at pH 9 and several
00:03:03.00 hours at pH 7, well about an hour or
00:03:07.00 something at pH 7. So, if you do a pH 9
00:03:10.00 reaction it's all over in an hour or
00:03:12.00 something, if you do a pH 7 reaction you
00:03:14.00 may want to leave it overnight, say at 4°.
00:03:16.00 Now, if you want to control the
00:03:19.00 stoichiometry, you don't want too much
00:03:21.00 label. If you overlabel a protein it may
00:03:23.00 quench the solution or be
00:03:25.00 non-fluorescent. With the Thiols, you
00:03:27.00 usually do this by stoichiometry, so you
00:03:29.00 add 1.5 moles per protein, or not too
00:03:32.00 much anyway. With the Amine reactive
00:03:34.00 probes, because they're hydrolyzing,
00:03:37.00 you're often controlling the reaction
00:03:39.00 not by stoichiometry, not by so much
00:03:42.00 so many moles per mole of protein but
00:03:44.00 rather by concentration. But in practice,
00:03:47.00 whether it's more stoichiometry limit, or
00:03:50.00 more concentration limited, depends on
00:03:51.00 the concentration of your protein and so
00:03:53.00 you may have to pilot it. The good news
00:03:57.00 about reacting tough proteins like
00:03:59.00 antibodies is it turns out they'll
00:04:04.00 accommodate a fairly wide range of
00:04:05.00 protein density so it's actually fairly
00:04:07.00 easy to get a good labeled antibody.
00:04:11.00 A few other practical points. Your protein
00:04:13.00 may have some essential cysteines or
00:04:15.00 lysines. I have spent half my life
00:04:16.00 working on the molecule tubulin and it
00:04:19.00 turns out if you take tubulin dimer form
00:04:22.00 in solution, it has highly reactive
00:04:24.00 cysteines and lysines that are essential
00:04:26.00 for its polymerization function and
00:04:28.00 there are some tricks for protecting
00:04:30.00 those. So, you can sometimes sterically
00:04:33.00 blockade essential cysteines or lysines
00:04:37.00 by having a high concentration of a
00:04:38.00 physiological ligand that blocks
00:04:40.00 access of the labeling reagent. So, for
00:04:43.00 example in the case of tubulin, you have
00:04:45.00 to label in the polymer. So, if the tubulins
00:04:46.00 polymerize into a microtubule
00:04:48.00 that actually protects these essential
00:04:50.00 groups. And sometimes with an enzyme you
00:04:52.00 can protect by having a high amount of
00:04:55.00 substrate that protects the active site.
00:04:57.00 I think my final practical point is
00:05:00.00 separating free probes from protein.
00:05:02.00 Traditionally you do this by molecular
00:05:03.00 size by dialysis, which is kind of slow,
00:05:05.00 or by gel permeation chromatography. With
00:05:09.00 these modern dies with the sulfonate
00:05:11.00 groups that works quite well. But if you
00:05:13.00 have a more sticky die it often will
00:05:14.00 stick to the protein and be hard to
00:05:16.00 separate. And it's useful for check how
00:05:18.00 much is left by chromatography on silica
00:05:21.00 gel. Increasingly, in my lab we like to use
00:05:24.00 an affinity based method labeling either
00:05:26.00 in solution, or first attaching the
00:05:28.00 protein and labeling it on a column. So
00:05:30.00 for example, with antibody labeling we're
00:05:32.00 often attaching the protein, to a protein,
00:05:35.00 an antibody to a protein A column,
00:05:37.00 labeling it on column, flushing away the
00:05:39.00 excess and then eluting the antibody.
00:05:41.00 That seems to work really well and give
00:05:43.00 us very little free die.

This Talk
Speaker: Tim Mitchison
Audience:
  • Student
  • Educators of Adv. Undergrad / Grad
  • Researcher
  • Educators
Recorded: July 2012
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Talk Overview

In this lecture about labeling proteins with fluorescent probes, Timothy Mitchison extends the lecture of Fluorescent Probes by providing more details and practical tips on how to covalently react fluorescent dye molecules to proteins.

Speaker Bio

Tim Mitchison

Tim Mitchison

Tim Mitchison completed his undergraduate degree in biochemisty at Oxford University before moving to the University of California, San Francisco to do his PhD with Marc Kirschner. After a brief post-doc at National Institute for Medical Research in London, Mitchison returned to UCSF as an assistant professor in 1988. In the late 1990s, he moved… Continue Reading

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