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Skipping Negative Stain

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Jessica Bruhn & Giovanna Scapin

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Skipping Negative Stain

For some complexes, direct to cryo-EM might be best.

Negative stain transmission electron microscopy (NS-TEM) analysis coupled with 2D classification can provide incredibly valuable information about aggregation state and conformational composition of a protein sample prior to vitrification.

But in negative stain the sample is diluted to 10-50 ug/ml prior to grid preparation – typically two orders of magnitude lower than concentrations used for cryo-EM grids (~0.5 - 4 mg/ml). Low concentration requirements sound great, especially for difficult to produce samples, but is it always a good thing?

Protein complexes stability depends on their dissociation constants (KDs), that for the simple case of a 1:1 reversible complex is defined as

Kd = [A][B]


Equilibrium at Negative Stain Concentrations

Even with a fairly high affinity complex, starting concentrations low enough will lead to dissociation.  For example, for a 200 kDa complex with an affinity of 10 nM and 1:1 stoichiometry only 75% of the complex is expected to remain intact at the concentrations typically used for negative stain grids (highlighted in gray) once equilibrium is reached; the mole fraction for the complex is only 61%, meaning that of all the types of particles in solution (protein A, protein B and the complex AB), only 61% are the particle of interest (complex AB). This may lead to confusing images and may make data processing and interpretation very difficult. This problem gets even worse with larger complexes and lower affinity interaction and complexes with more complicated stoichiometries and multiple KDs.

But it is not a lost cause…for complexes between a protein and a small molecule, simply increasing the ratio of small molecule to protein (5-10 fold excess) will shift the equilibrium in favor of almost exclusive formation of the complex. For protein-protein complexes, useful negative stain data can be obtained by properly calculating the sample dilution, or if the samples dilution is done immediately prior to grid preparation (if the complex has a slow koff).  As a last option, skip negative stain entirely and go directly into ice.  Screening a vitrified sample is more time and sample consuming, but it might be the best option.

At NIS, we pride ourselves on working with our clients to come up with strategies the have a high chance of success. Schedule a consultation with one of our scientists to discuss project feasibility and the best way to get started.

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