Recombinant protein expression in HeK cells?

When you are not observing any protein expression on a Western blot after transfection, several factors could contribute to this issue. Here are some common considerations and troubleshooting steps:

Transfection efficiency: Although you observed good transfection efficiency with the GFP vector, it is important to verify the transfection efficiency of your protein of interest (POI). Ensure that your POI construct is successfully transfected into the cells by using appropriate controls, such as a positive control vector expressing a known protein that can be detected on a Western blot.

Expression kinetics: The expression kinetics of your protein may differ from that of GFP. Some proteins may require more time to reach detectable levels after transfection. Consider extending the post-transfection incubation time to allow sufficient protein expression. It is advisable to perform a time course experiment to determine the optimal time point for protein detection.

Promoter strength: Confirm that the promoter driving the expression of your protein is active in HEK293T cells. Different promoters have varying strengths, and it's possible that the promoter you are using may not be efficient in driving gene expression in this specific cell line. Consider using a stronger promoter or optimizing the promoter sequence to enhance expression.

Proper protein extraction: Ensure that you are using an appropriate lysis buffer and extraction method to solubilize and extract the protein from the transfected cells. Different proteins require different lysis buffers, detergents, and extraction conditions. Make sure your lysis buffer is compatible with your protein of interest.

Detection antibodies: Confirm that the antibodies used for protein detection are suitable and specific for your protein. Ensure that the antibodies are validated and have been successfully used for Western blotting in previous studies. Optimize the antibody concentration and incubation time to maximize detection sensitivity.

Protein stability: Some proteins may have short half-lives or may be prone to degradation. Ensure that you are taking necessary steps to maintain protein stability, such as using protease inhibitors in the lysis buffer, performing the experiments at lower temperatures, or adding protein stabilization agents.

Sample loading and gel electrophoresis: Verify that you are loading an appropriate amount of protein lysate on the gel for Western blot analysis. Use a loading control protein, such as GAPDH or β-actin, to ensure equal protein loading across the samples. Optimize the gel percentage and electrophoresis conditions to ensure proper separation of proteins.

Blotting conditions: Optimize the transfer conditions, including transfer buffer composition, transfer time, and transfer method (wet or semi-dry), to ensure efficient transfer of proteins from the gel to the membrane. Verify that the proteins are successfully transferred by using reversible staining methods or by Ponceau S staining before antibody incubation.

Detection method: Evaluate the sensitivity of your detection method. If you are using chemiluminescent detection, ensure that the substrate is fresh and that the exposure time is sufficient to capture weak signals. Consider using more sensitive detection methods, such as enhanced chemiluminescence (ECL) or fluorescent detection.

By carefully considering and troubleshooting these factors, you can identify the potential cause of the issue and optimize your experimental conditions to detect the expression of your protein of interest.

Manuele Martinelli

Dear Pawan Yadav

just some questions:

1) did you add the Kozac sequence (gccgcc or gccacc) before the ATG starting codon in your insert? Since the pcdna vectors do not contain it and you have to add in your insert. Kozac sequence is similar to the RBS binding site for bacteria, its present is essential.

2) Would you like to express the protein intra-cellular or secrete it into the culture medium? And if is the second case, did you ad an N-terminal leader peptide to your sequence?

best regards

Manuele

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