You're encountering a frustrating but surprisingly common issue: strong GFP fluorescence but no corresponding GFP band in western blot, despite your Actin control working well. Since your workflow appears technically sound, the issue likely lies at the interface of protein detection, degradation, or epitope masking. Here are the most plausible causes and solutions, customized to your system:

🔍 1. Epitope Masking or Conformational Interference

Even though GFP fluoresces (indicating it's folded properly), the epitope recognized by the anti-GFP antibody might be conformationally hidden in your fusion protein. Some anti-GFP antibodies (especially monoclonals) only recognize denatured or specific linear epitopes, and if your fusion protein alters this structure, the antibody may fail to bind in western blot conditions.

✅ Solution:

• Switch to a different anti-GFP antibody, ideally one validated for WB of fusion proteins (e.g., Thermo Fisher A-11122 or JL-8 from Takara).
• Test a polyclonal anti-GFP antibody, which may recognize multiple epitopes.
• Consider tagging your protein with an N-terminal or C-terminal epitope tag (like HA, FLAG, or Myc) and probing with anti-tag antibodies.

🧪 2. GFP Degradation or Cleavage in Planta

Your protein may be expressed, fluorescing in planta, but undergoing post-translational cleavage or degradation, especially by plant proteases. This could remove GFP or render it undetectable by western blot.

✅ Solution:

• Run a shorter post-infiltration harvest timepoint (e.g., 48–60 hpi) to reduce degradation.
• Include more potent protease inhibitors, or add fresh PMSF just before lysis (it degrades quickly).
• Analyze smaller bands (
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Clearly your WB itself is OK. How did you check your "clear fluorescence"? Was it much more intense than your untransfected control? All cells fluoresce a bit, so make the difference is obvious. Did you sequence your fusion construct? If your GFP is a c-terminal fusion, a misframe could explain your issue. If you do have a strong signal by IF, then the problem might be that your protein is insoluble in your lysis buffer. You could check for fluorescence in your post lysis pellet. Alternatively, your protein might be unstable. I am not familiar with plant systems but you could try denaturing your samples right away, if possible. This would address both issues. Since you use Tubulin as a housekeeper, BCA is not essential (once you know the typical amount of proteins in you samples). Finally, it's good practice to perform a total protein stain on your samples. This will ensure that your protein load is OK throughout, not just where your housekeeper migrates. Good luck!

Sébastien Soubeyrand Thanks for your response! The signals (expressions) were checked using a Zeiss confocal microscope, and the fluorescence looked average—not too weak, not too strong.

Yes, I also did sequencing, and it was correct. The GFP is fused at the C-terminus.

Also, since the actin bands show up in my Western blot, that means the protein extraction worked fine, right?

i did not check the pellet for fluorescence but I will check this time when I extract protein.

Actin is readily soluble, you POI may not be so soluble. Is your POI hydrophobic? Where is your signal by confocal? If diffuse, should be readily extractable. If not, may be bound to cell structures and harder to extract.

Sébastien Soubeyrand the signal is seen within the cell in the nucleus