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Ugh. Did an immunofluorescence experiment last weekend, forgot to vortex both my primary and my secondary antibody solutions.

And my final result looks dimmer than it should be. Is it possible that not vortexing contributed to this?

I guess the question is, is vortexing really necessary? It would come down to the fluid dynamics. How long does it take for a drop of antibody to reach equilibrium in a 1000µL Eppendorf tube? Is it really more than just a few seconds? Is vortexing really necessary?

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It depends on how much time elapsed between adding the concentrated antibodies to the diluting solution and adding the diluted antibodies to the slide. If it was just a couple of seconds, then I can abolutely see there being a difference. Even if it was longer - say 30-90 seconds, even - there potentially could be antibody gradients in the solution without vortexing. There are a few reasons why. First, many unconjugated primary antibodies that are not intended just for immunostaining are provided in a glycerol solution, with concentrations ranging from 10% or so up to 50% or more, sometimes in addition to a carrier protein like BSA (I used to work for a well-known antibody company, and theirs are in 50% glycerol with 10 µg - 2 mg BSA per ml, depending on the antibody type). The glycerol prevents the solution from freezing solid at -20°C, eliminating ice crystal formation, protecting the antibody protein from degradation and allowing for longer storage times without loss of activity. As a result, however, the solution takes longer to solubilize in PBS or whatever diluent you're using on its own, with no additional mechanical activity.

On the other hand, even if the primary (or secondary) antibody is simply formulated in PBS, the equal diffusion of the proteins to all parts of the tube can take some time. As an experiment, put 1 ml of PBS in an Eppendorf tube, then add perhaps 10-50 µl of a dark dye, such as methylene blue, bromphenol blue, or Trypan blue. Watch it dissociate into the solution over time against a solid white background like a piece of paper. While none of these have the exact same solubility profile as antibodies, it's good enough to show you what happens over the first 30 seconds or so until you can't distinguish the dye anymore.

So, always vortex, even if it's at a relatively slow speed, for a couple of seconds to ensure everything is evenly distributed.

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    $\begingroup$ I assume that a small molecule dye will probably diffuse faster than a large antibody. So if a dye takes time to fill the entire volume, an antibody likely needs more time. $\endgroup$ – user137 Jun 27 '16 at 0:20
  • $\begingroup$ @user137 that's exactly my point. $\endgroup$ – MattDMo Jun 27 '16 at 2:21
  • $\begingroup$ i was taught never to vortex proteins that i wanted to do something useful, but the even mixing (by pipetting or inversion or extended nutation or whatever) of a solution is pretty much required for us to say anything meaningful about any biochemical reaction. In conclusion, the @MattDMO answer is right allowing for local variation in methodological customs. $\endgroup$ – Maximilian Press Jun 27 '16 at 6:03
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    $\begingroup$ @MaximilianPress you were taught incorrectly. The only protein I don't vortex is DNase I, used for digesting DNA on an RNA prep spin column - and only because the directions say to not vortex it. Proteins are in general very structurally stable, and a weak force such as vortexing won't damage them one bit, I promise. I have been working with proteins for nearly 20 years, and I've never met a biochemist who said that proteins in general, or antibodies specifically, shouldn't be vortexed. Now, if you're doing native conformation co-IPs, for example, you can of course be a little more (...) $\endgroup$ – MattDMo Jun 27 '16 at 11:57
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    $\begingroup$ (...) delicate than when working with an antibody for Western blotting or immunofluorescence, but even there the danger is more heat than physical force. Antibodies are incredibly tough and resilient, can often work in a wide range of chemical environments, and in general only need to be treated specially when they're conjugated to light-sensitive fluorochromes. Yes, you don't want bubbles to necessarily form in your solution, but again that's only when you're working in a conformationally-sensitive assay. $\endgroup$ – MattDMo Jun 27 '16 at 12:02

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