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Proteins in glycerol and nano-injection

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Maria Milla

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My protein solution contains glycerol: can I inject it to the cells without compromising the viability?

Proteins come often diluted in buffers containing glycerol: either for storage at -20°C (1), for stabilization or prevention of protein aggregation (2, 3). The amount of glycerol depends mainly on the protein of interest. For protein preparations and storing at -80°C, 10-15% of glycerol is enough to prevent protein degradation. For storage at -20°C, higher concentrations of glycerol are normally required (ca 50%). 

Although some studies have analyzed the behavior of cell cultures in presence of glycerol, suggesting that the presence of this compound at a high concentration can reduce cell proliferation (4), we do not think the presence of glycerol can reduce the viability of the cells for nano-injection of protein solutions for the following reasons: 

- Some protein stocks contain up to 50% of glycerol, but this solution is too concentrated for a direct injection into the cell. As example, Cas9 protein stock solution offered by some companies, contains 50% of glycerol, but it must be diluted at least 5 times to adjust the concentration. Therefore, the final concentration of glycerol will be lower than 10%, which is supposed to be safe for the cell (5). 

- Several studies have determined that glycerol is diffused out of the cell in order to control the homeostatic levels within the cell (6), so after the increase of glycerol inside the cell, this can be cleared from the cell. 

- Moreover, some studies have even used glycerol as an agent to increase the contrast of the cell for phase-contrast microscopy. For example, Rommel et al (7) injected solutions of 50% glycerol into cells with this purpose: cell membrane maintained its integrity and cell viability was not affected. 


References
(1) Simpson RJ (2010) Stabilization of proteins for storage. Cold Spring Harbor Protocols, 5 doi: 10.1101/pdb.top79
(2) Gekko K, Timasheff SN (1981) Mechanism of protein stabilization by glycerol: preferential hydration in glycerol-water mixtures. Biochemistry, 20 (16): 4667-4676 doi: 10.1021/bi00519a023
(3) Vagenende V, Yap MGS, Trout BL (2009) Mechanisms of Protein Stabilization and Prevention of Protein Aggregation by Glycerol. Biochemistry, 48 (46) 11084-11096 doi: 10.1021/bi900649t
(4) Wiebe JP, Dinsdale CJ (1991) Inhibition of cell proliferation by glycerol. Life Sciences, 48(16) 1511-1517
(5) Davidson AF, Glasscock C, McClanahan DR, Benson JD, Higgins AZ (2015) Toxicity Minimized Cryoprotectant Addition and Removal Procedures for Adherent Endothelial Cells. PloS ONE, 10(11) e0142828 doi: 10.1371/journal.pone.0142828
(6) Yang NJ, Hinner MJ (2015) Getting across the cell membrane: an overview for small molecules, peptides, and proteins. Methods in Molecular Biology, 1266: 29-53 doi: 10.1007/978-1-4939-2272-7_3
(7) Rommel CE, Dierker C, Schmidt L, Przibilla S, von Bally G, Kemper B, Schnekenburger J (2010) Contrast-enhanced digital holographic imaging of cellular structures by manipulating the intracellular refractive index. Journal of Biomedical Optics, 15 (4): 041509. doi: 10.1117/1.3449567

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