Hi this may sound like a really stupid question but im totally new to lab work. If i purchase plasmids like the one below,
SYNPO (GFP-tagged) - Human synaptopodin (SYNPO), transcript variant 3, (10ug)
and intend to detect the protein localization after transfection, i dont need any primary or secondary antibodies? so how is this thing visualized?
i usually use Immunofluorescence steps like fixing, permeabilization, primary antibody incubation, secondary incubation etc.
Hi, GFP is actually fluorescent by itself, you'll just need the right filters to observe it with a microcope. If you have too much background then you can go for an immunostaining against GFP to increase the signal.
Before transfection, you need to optimize the selection concentration (antibiotic concentration, see on your plasmid map and refers other papers).
18-48hrs after transfection you can see GFP expression under fluorescent microscope. This depends on plasmid. It is unnecessary to conduct ICC.
Then go for selection to get purified-transfected cells. Long time without selection or long time with selection , GFP level in both cases is decreased.
If you wanna define the localization of new proteins whether in cytoplasm or nucleus..., you can try to observe them under confocal microscope.
I hope that helps
if your transfection is success, your cells will express GFP protein and we can directly see the green fluorescent colour cells under fluorescent microscope
Sometimes it can happen that GFP signal is weak, then you can use an immunofluorscence staining with an antibody against GFP. You need to fix cells
Following transfection and if your GFP is exprtessed you can fix and follow with another anti-actin antibody using of course a secondary with a fluorophome emmiting at a different wave length
In your plasmid, the stop codon is absent in between your protein and GFP tag in order to get protein tagged with GFP. GFP excitation is at 488nm which is the green pass filter. So depending upon the transfection reagent you are using, incubation period may differ starting from a couple of hours to 72 hours in some cases. After transfection, you can directly look for GFP expression in green filter in particular compartments like the membrane, cytoplasm, nucleus or any other organelle for that matter or you can even goat, rabbit, mouse.. raised primary antibodies against GFP or your over-expressed protein and use Alexa flour 488, 594, 647nm secondary anti-goat, anti-rabbit or anti-mouse antibody based on the availability of specific filters in the instrument which you would use for imaging.
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