Dear all,
I plan to test the internalization of an antibody on a panel of human cells.
However, the supplier added sodium azide to it, which inhibits internalization.
The final NaN3 concentration in the wells will be 2 micromolars.
Any idea whether this range of concentration affects already internalization/intracellular trafficking ?
Thank you,
Bets regards.
Dear Benjamin,
Please You look at the following articles:
Quantifying Nanoparticle Internalization using a High Throughput Internalization Assay
https://minerva-access.unimelb.edu.au/bitstream/handle/11343/282777/11095_2016_1984_ReferencePDF.pdf?sequence=1&isAllowed=y
NaN3 (120 mM) was used to inhibit all energy-dependent internalization pathways, and resulted in a strong decrease in NP internalization in both cell types (87% in 3T3; 82% in CEM; Fig. 6b and S9) (page 19)
The effect of particle design on cellular internalization pathways
https://www.pnas.org/content/pnas/105/33/11613.full.pdf
Fig. 6. Probing the mechanisms of cellular internalization by using inhibitors of endocytosis. HeLa cells were incubated with the indicated inhibitors in the graph as outlined in the experimental methods. Percent internalization was normalized to particle internalization in the absence of inhibitors.
Hello Benjamin Tschumi
I would suggest that you remove sodium azide from the antibody solution. It would be difficult to decide on the concentration of NaN3 since you will be working on a panel of human cells. You could make use of methods like dialysis, desalting or antibody purification kits. The protocol is provided in the link below.
https://www.antibody-creativebiolabs.com/sodium-azide-removal-protocol.htm#:~:text=A%20dialysis%20unit%20can%20be,sodium%20azide%20is%2065%20Da.
Best.
Hi, thank you for your replies.
I think I will completely remove NaN3 as the inhibiting concentration is unknown and can vary depending on the cell line.
Best regards,
Benjamin
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