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Transient transfection protocol for HEK293T cells

Protocol created by Lorena Maestre - Monoclonal Antibodies Unit, Centro Nacional de Investigaciones Oncológicas

Transfected cells are important in investigating the specificity of antibodies and also permit studies on the regulation and function of proteins. Transient gene expression is the temporary expression of genes resulting from introducing foreign genetic material into a eukaryotic cell. A variety of techniques, e.g. chemical and biological methods, can be used to transfect cells.

Schematic representation transient transfection

Plasmids containing a reporter or 'tag' gene (e.g. MYC or GST) in addition to the DNA encoding the target antigen of interest are commonly used. The high copy number of the DNA plasmid results in the high expression of the target antigen. Results can be seen after 24 to 96 hours of culture but the foreign genetic material does not integrate into the chromosomes and so the target antigen is lost during prolonged culture. siRNAs, miRNAs and mRNAs can also be used for transient transfections.

A range of different eukaryotic cell lines (e.g. HeLA, COS and HEK293T) can be used and the success rate and duration of target antigen expression depends upon the genetic material introduced and the type of eukaryotic cells used. In our experience, HEK293T cells are easily transfected and the percentage of transfection can be around 80% by using the present protocol.

Cell line:

Culture medium and equipment:


Transfected cells expressing your target antigen are valuable tools in antibody validation, especially if you want to check antibody cross reactivity with other proteins that share high homology. The cells can then be used to make cytospins for immunocytochemical labelling or cell pellets for Western blotting studies.

Validation of the reactivity and specificity of the anti-CD85A mAb FRAS92B

Figure 1. Validation of the reactivity and specificity of the anti-CD85A mAb FRAS92B. A) Immunocytochemical staining for CD85 family members on cytocentrifuge preparations using FRAS92B mAb. Staining of antibody FRAS92B was observed in HEK-CD85A-Myc transfectants but not in HEK-CD85B, CD85C, CD85D, CD85E, CD85F, CD85G, CD85H, CD85I, CD85J and CD85K. The anti-Myc, anti-GFP and anti-V5 mAbs were used to confirm transfection efficiency. B) The specificity of the antibody was also confirmed by the Western blotting technique using the transfected cell extracts. A strong band corresponding to CD85A was detected only in the HEK-CD85A cell extract.