We have prepared a construct using GFP sequences in middle. Although sequencing analysis confirmed the positive clone, but transfection results shows no signals after various attempts. The positive control clone shows fluorescence under the microscope but not the same results for target construct. What might be the reason for no fluorescence. Kindly guide with your valuable suggestions...
Thank you in advance
Is the fusion protein expressed and full length (Western blot) - if yes, then it probably is not folding or cyclizing properly. This post-translational process is inefficient but essential, with a t1/2 of several hours or more, depending on the FP. Flanking peptide regions can inhibit this. Sometimes, a proline ( to separate protein domains) and then several serine/glycine residues flanking the FP to act as a flexible spacing linker can help.
If no protein, did you include a Kozak sequence (ribosome binding site) in front of the initiating ATG codon?
If the plasmid sequence is right and quality is good so i think there may be some misfolding in GFP.
What is the protein and the expression system? If heterologous expression, please see if post-translational modification is involved?
Hi there,
What is the promoter used? Is it the same than in the control cell? If yes then he protein of interest might provoke fusion aggregation. Did you check at the RNA level?
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