3 replies to this topic

[porfirion]

HI,
I am doing transfection of CHO cells with some proteins, and sort of NEED them to be on the plasma membrane (they are transmembrane proteins) to be shed by proteases, however each time I can see them in Golgi network, but not reach surface, Any idea how to do this, I using Fugene HD products with standard protocol.
Help!!!!!!!!!!!!!!!!!!!!1

[wirly]

porfirion, on Nov 5 2009, 05:17 AM, said:

HI,
I am doing transfection of CHO cells with some proteins, and sort of NEED them to be on the plasma membrane (they are transmembrane proteins) to be shed by proteases, however each time I can see them in Golgi network, but not reach surface, Any idea how to do this, I using Fugene HD products with standard protocol.
Help!!!!!!!!!!!!!!!!!!!!1

You didn't remove the signal peptide form the N-term when cloning into your expression vector, did you?  The trasfection reagent will not make a difference.

more details?

[porfirion]

The protein was purchased as a full-lenght expression plasmid so no, I didn`t remove anything.

[wirly]

porfirion, on Nov 11 2009, 08:12 AM, said:

The protein was purchased as a full-lenght expression plasmid so no, I didn`t remove anything.

Do they naturally traffic to the surface?  Do they have a Golgi retention signals of some sort?  Are they surface-cycling proteins?  Are you clipping them at the surface with a reagent or are they naturally clipped?  If the latter, are they being clipped from the surface so efficiently that you cannot detect them?  If this is the case, include inhibitors and possibly ice-cold incubations during all staining steps.  How are you detecting them at surface and internally?

more information is more helpful than less