This kind of situation can affect different issues such as follows;

You can read about the following strategies a little more and try them instead of what you have followed.

• Use agents like chloroquine, polyethyleneimine (PEI), or lipofectamine to promote endosomal escape. These compounds help release proteins from endosomes into the cytoplasm.
• Confirm that the C-terminal NLS is exposed and functional. If there's a risk of folding masking the NLS, consider alternative designs, such as an N-terminal NLS. Alternatively, tag the protein with a cell-penetrating peptide (e.g., TAT or R9) to enhance nuclear delivery.
• Allow longer recovery times (e.g., 12–24 hours) to ensure proper localization and expression. Include nuclear import facilitators such as Ran-GTP or importins to improve the efficiency of nuclear import.
• Perform live-cell imaging soon after electroporation (e.g., 2–4 hours post-treatment) to check for transient nuclear localization that might be lost after extended recovery.
• Consider using microinjection, lipid-based transfection, or nanoparticle-based delivery, which might bypass endosomal entrapment.
• Test different buffer compositions to reduce endosomal uptake. Reduce protein concentration during electroporation to minimize aggregation. Optimize the electroporation voltage and pulse duration for HeLa cells specifically.
• Use a fluorescently labeled protein with a well-characterized NLS (e.g., GFP-NLS) to validate your electroporation protocol. Confirm the integrity and activity of the labeled Cy5-protein using in vitro assays.

Dear Theja,

I sincerely appreciate your response and suggestions.

I tried to incubate my Cy5-labeled protein with live cells directly overnight, and I observed that the protein was trapped in the endosomes and could not escape. This is why I decided to try electroporation as an alternative. Based on my understanding, most of the proteins should not remain in the endosomes after electroporation, so the phenomenon I observed was quite surprising.

• "Perform live-cell imaging soon after electroporation". I plan to try this first because I think the proteins may be excluded from the nucleus over time, so observing the cells immediately might let me know what occurs right after electroporation.
• "tag the protein with a cell-penetrating peptide (e.g., TAT or R9)". I also plan to construct a plasmid containing N-terminal TAT. I hope this sequence can help my protein escape from endosomes.
• "chloroquine, polyethyleneimine (PEI), lipofectamine, lipid-based transfection, and nanoparticle-based delivery". I need to check more papers to identify the most suitable approach for my proteins.
• "longer recovery times". As I said, I tried to incubate the protein with live cells overnight, and proteins were still trapped in endosomes. I also suspect that the protein might degrade over time.
• "Reduce protein concentration during electroporation to minimize aggregation". I have tested lower protein concentration and I observed the same results. My protein is not particularly stable, and although I have optimized a buffer condition to maintain its stability, I am uncertain whether this condition is effective during electroporation. For electroporation, I resuspended my cells with Opti-MEM.
• "Use a fluorescently labeled protein with a well-characterized NLS (e.g., GFP-NLS) to validate your electroporation protocol". I greatly appreciate your suggestion because I am looking for something can be used as a positive control. I will purify this protein.

By the way, the isoelectric point of my protein is above 10. Do you think this could affect its ability to enter the nucleus?

Thank you once again for your advice!

Best,

Liang

Dear Liang,

Your observations and planned experiments are well thought out! and you are welcome.

Yeah, the Isoelectric point may affect the results. A protein positively charged at physiological pH and with a high Isoelectric point (pI) is said to be strongly basic. This could improve interactions with nuclear import machinery and negatively charged cell membranes, but it could also lead to aggregation or impede diffusion because of strong charge-based interactions. Therefore, ensure the buffer used for electroporation and incubation maintains physiological ionic strength to mitigate aggregation. Avoid using excessive salt, which could destabilize cells during electroporation.

The stability of your protein during electroporation is critical. Opti-MEM is widely used, but if stability is a concern, consider supplementing with stabilizing agents like glycerol, BSA, or small amounts of reducing agents (e.g., DTT, if compatible).

Best Regards!

Theja

Theja Virajini

Dear Theja,

I truly appreciate your response! Your knowledge has provided valuable insights for my experiment. I now have clear ideas on how to improve my protein delivery. Thank you very much!

Happy New Year!

Best,

Liang

Theja Virajini

Dear Theja,

It has been two months, and my electroporation now works successfully. I truly appreciate your expertise!

Best,

Jingchang