I've been expressing the SEP-tagged GluA1 and GluA2 in organotypic hippocampal slices but am not seeing anything resembling surface AMPARs with either subunit. The GFP signal is homogenous throughout the cell and there does not appear to be any difference between SEP-GluA1 and SEP-GluA2. I have looked at their expression at 24, 36, 48 and 72 hours after transfection. I'm using biolistic transfection and imaging live cells. I've checked the pH of my solutions and sequenced the constructs. I've expressed the SEP constructs in the presence of td-tomato, as a filler. Most often, I'm seeing approximately equal levels of GFP and td-tomato signals (rather than GFP>td-tomato at the plasma membrane as expected). I've also looked at the SEP constructs alone (no td-tomato) and saw no difference. Nuclear exclusion of the SEP signal is not consistent. From what I understand, these constructs are generally very reliable so I'm not sure why I'm having this problem. Does anyone have any suggestions? Thanks for the help.
How exactly does the vector look like? If you cloned the tag C-terminally, you might cover a sorting signal so the recombinant protein is not sorted into the secretory pathway but expressed cytosolically. Which promoter do you use? If the protein is overexpressed you might get protein just everywhere so the strong intracellular signal covers up the rather weak signal in the membrane. I have never worked With SEP tags, are they known to alter the subcellular localization? E.g. old GFP variants had a nuclear localization signal.
Good Luck! Tilo
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