HEPES transfection - another cell transfection method

攝影師:HAMID ELBAZ: https://www.pexels.com/zh-tw/photo/225869/

Article Author: GuoChian Li / R&D of Fine Chemical Division


Efficient transfection of functional proteins into cells is a common means of regulating cells, enabling the exploration of protein-protein interactions and intracellular protein transport.


Common protein transfection methods fall into two categories - chemical and physical:

  1. ChemicalUsing macromolecular carriers to carry proteins into cells.
  2. Physical electroporationTo temporarily perforate the cell membrane to allow proteins to enter the cells.


In addition to the above two methods, this paper proposes to use HEPES for protein transfection (Figure 1). The transfection efficiency using HEPES (< 50 mM HEPES) is not inferior to commercially available transfection reagents (Ex.PULSin, ProteoJuice, Pro-Ject, Xfect, BioPORTER QuickEase) on experimental ovarian cancer cells (MDAH 2774), and the cell viability and cytotoxicity were not significantly affected.


Commercially available transfection reagents are more expensive than using HEPES transfection method. This technology has been used in various cell culture media for a long time, so there is no doubt about safety and stability.

Figure 1 Flow chart of protein transfection using HEPES (from literature)


HEPES transfection conditions


Different types of protein samples and cells require different HEPES concentrations and mixing time, and the optimal HEPES transfection method needs to be adjusted through experiments.


It is recommended to use the initial conditions of mixing 1 mg of protein sample with 50 ml of 20 mM HEPES solution for 15 minutes before cell culture and protein transfection. In addition, the use of Opti-MEM (reduced serum medium) and HEPES transfection method for cell culture is optimized.


Principle of HEPES transfection method


Protein samples are multiplex, such as:

  1. Antibodies (Alexa Fluor 488, etc.), molecular weight about 150 kDa
  2. Recombinant protein (rhSTIP1) with a molecular weight of about 66 kDa
  3. FLAG-tagged-Rve peptide, molecular weight less than 5 kDa


Exploring how HEPES transfects protein into cells through differences in sample properties (EX. structure, composition, molecular weight, and charge).



In cell experiments, it was found that cells facilitate the transfection of proteins through three types of endocytosis:

  1. Macromolecular substances (200 ~ 500 nm) through macropinocytosis and caveolae-dependent endocytosis, and
  2. Small molecules (< 200 nm) via clathrin-dependent endocytosis.


In the same cell experiment, no transfection effect was found in another commonly used Good’s buffer - Tris-HCl. NMR experiments further found that the diffusion coefficient of HEPES solution would be decreased by 25% after adding recombinant protein. The zwitterionic nature of HEPES neutralizes the surface charge of protein samples is the evidence.


Cell types suitable for HEPES transfection method

  1. Breast cancer cells (MCF7)
  2. Cervical cancer cells (HeLa),
  3. Colon cancer cells (HT-29)
  4. Endometrial cancer cells (ARK2, RL952)
  5. Glioblastoma cells (U87)
  6. Hepatocellular carcinoma (HepG2)
  7. Lung adenocarcinoma cells (CL1-0)
  8. Erythroleukemia cells (HEL)
  9. Mouse Ovarian Cancer Cells (MOSEC),
  10. Nasopharyngeal carcinoma cells (NPC-BM1)
  11. Non-Hodgkin's Lymphoma (Toledo)
  12. Ovarian cancer cells (MDAH2774 and SKOV3),
  13. Pancreatic Cancer Cells (BxPC3)
  14. Renal cell adenocarcinoma cell (786-O)
  15. Sarcoma cancer cells (SK-LMS-1)






Utilization of HEPES for enhancing protein transfection into mammalian cells (https://doi.org/10.1016/j.omtm.2018.12.005)

Further Reading: Why use HEPES?
Further Reading:HEPES handling and storage tips that you must know
Further Reading:HEPES VS PBS (phosphate buffered saline)

Release date:2022.06.22