Failed to express a protein - (Aug/15/2006 )
I can´t find my protein after transfection. I have tried to do it with another gene in the same vector and I can see that one on western blot but not mine. I have tried proteosome inhibitors and codon optimasation. Has anyone any idea of what could be wrong?
Have u checked antibodies?
R u sure that the protein is easily extractable in ur lysis buffer?
I can´t find my protein after transfection. I have tried to do it with another gene in the same vector and I can see that one on western blot but not mine. I have tried proteosome inhibitors and codon optimasation. Has anyone any idea of what could be wrong?
Have u checked antibodies?
R u sure that the protein is easily extractable in ur lysis buffer?
I have 2 different antibodies that work on the pos. control and the protein should be extractable in the buffer.
If everything seems fine incld. tranfection and anitbodies, I would suggest u have a closer look at the expression vector and the transgene sequence. May b the trangene is not being expressed.
is it transient or stable transfection?
the solubility of protein in extraction buffer may change if your expression vector drive too big quantity of protein in the cell. I mean a standard quantity gives no pb, but protein goes to inclusion bodies or sthg similar when too concentrated in the cell.
I've read that you can directly boil your cells in loading buffer and goes for a gel. That could be a first try to detec protein expression...
Yeh, if you want to just to check for protein expression on a quick WB then just add 1 X Laemmli sample buffer to the well, boil for 5 min and load onto a gel. It will be very viscous when you first collect them (due to DNA) but after you boil the sample, it should be fine. As a guide, i add 400ul per six well (~80% confluent) and run between 5 and 40ul onto a gel.
This method eliminates any issues regarding solubility and will tell you whether you have protein expression or not.