Hi Cristhina Jaramillo

The more information the clearer answer you would have.

1. Is your protein a nucleic protein, cytoplasmic protein or other?

2. What ratio TR-reagent/Plasmid have you used?

3. The number of cells per wells?

4. How long you wait to observe the efficiency?

5. In case of lipofectamine plus what is the ratio of (L3K/P3000) and (P3000/Plasmid)?

All the best...

Mohamed Khashan

My protein is a membrane receptor, transfected at 60-70% confluence.

The lipofectamine 2000/DNA ratio is 1:2, lipofectamine plus is 1:3 and PEI is also 1:3

I observed my transfection at 24 hours and the protein was still not expressed, at 48 hours post-transfection and there is a low expression and at 72 hours, it was further reduced.

Thanks for your help

Thanks for theses information Cristhina Jaramillo ,

According to a publication, in case of SH-SY5Y cell line, L3K is the most efficient one and the most toxic reagent.

After thawing your cells, subculture them at least one time then seed them in a high density (400k for 6 well-plate), leave them for one day.

Refresh the medium (In our lab we refreshed with DMEM one hour before transfection, others prefer to add Opti-MEM one hour before transfection. So you can optimize your one).

Prepare two tubes (1. 125 ul Opti-MEM + 5 ul of P3000 and 2.5 ug (you can use different amounts I used 2.5ug for 250k and got ~1.7 fold) of the plasmid) (2. 125 ul Opti-MEM + 7.5 ul of L3K).

Add the 2nd tube to the first tube vortex for 10 sec and incubate at room temp for 20 min.

Add the mixture over cells drop by drop and swirl the plate briefly.

You can refresh after 6 hours to reduce the toxicity (some reported that this can reduce the efficiency and others didn't, so again you can optimize that according to your experiment).

Of note, sometimes GFP can be broken down by disrupting the medium PH so it is possible to have a misleading result. It is better to detect the expression by RT-qPCR after not less than 48 hours.

In our lab we transfected two plasmids, one for nuclear protein and the other for a cytoplasmic protein and we got (10% and 20% efficiency respectively according to FACS) and ~1.7 fold by RT-qPCR.

If you have a reach to RNAiMAX, I can also provide you with a helpful protocol I performed and got ~3.5 fold expression.

All the best