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I am running an immunoassay using biotinylated antibodies and have noticed a decreasing trend from duplicate assay runs (completed within minutes of each other) from the same vial.

In between each run:

  1. I switch to a new low protein binding pipette tip.
  2. I vortex the biotinylated antibody in solution in a low protein binding vial.
  3. Then I prewet the pipette tip and deliver the reagent.

Note that degradation seems unlikely.

Any ideas on what may cause this? Could it be vortexing or shearing during pipette tip mixing?

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  • $\begingroup$ What kind of assay is this? i.e. ELISA, etc.? $\endgroup$ – Steven T. Snyder Feb 25 '14 at 18:32
  • $\begingroup$ Its a sandwich ELISA assay. $\endgroup$ – CCovey Feb 25 '14 at 20:05
  • $\begingroup$ 1) Was there any foam? 2) Is there any reason to think that solutes will settle, or is there condensation on the top of the tube, or has the reagent been frozen? If not, there might not be any reason to even mix the reagent. $\endgroup$ – Karsten Theis Aug 24 at 21:45
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I never worked in the field but applied mechanical stress (French Press, sonication) while extracting chlorophyll a from cyanobacteria.

Anyway, a cursory search furnished some sources like this, where vigorous vortexing of antibody samples is advised against. Instead, gentle or pulse vortexing or simple spinning is suggested.

But please take this with a grain of salt since I'm not really familiar with the standard protocols here.

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It is good practice to avoid vortex mixing any protein solutions, especially vigorously. Vigorous vortex mixing can produce foaming and proteins can denature (unfold and lose activity) when they are present at an air/liquid interface (as in a foam). This is because the hydrophobic regions of the protein will want to be in the air region and the hydrophilic regions will want to be in solution, and so unfolding may occur.

In practice, antibodies are usually quite stable as proteins go and you can often vortex them without much negative effect to your experiment. That said, if you routinely vortex proteins in the lab and come across a less stable one you will suffer degradation. Since you will not always know in advance which proteins will be OK to vortex, it is best practice to mix gently using a pipetman (again avoid introducing air that creates a foam) or inversion, or other method. If you must vortex, then be gentle with short pulses, avoiding foaming.

I have purchased some antibodies where the datasheet specifically says not to vortex mix.

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