detecting lacZ with anti-FITC - (Dec/01/2011 )
Hi everyone !
I am working with GeneSwitch system (invitrogen). After establishing stable lines with pSwitch vector, I would like to check if they work properly. With the kit they provide pGene/LacZ/V5/His positive control. Recently I have tried to transient transfect my cells, induce the expression and immunostain them with anti-V5-FITC (I did methanol fixation). Unfortunately there was no expression comparing to negative control.
Do you maybe know if the methanol fixation is the proper method for this kind of protein ? What else could went wrong ?
With best regards !
It could be any one of many things in the entire procedure that is the problem. Have you been able to successfully transfect plasmids into this cell line before? Have you tried to stain for lacZ ( using ONPG or IPTG) to see if the lacZ is being expressed or check for its presence on a western? Did you try to optimise the transfection at all? Did you check that the plasmid is what you think it is?
Have you successfully done immunostaining before? How old are your antibodies? which of the many procedures are you using (i.e. post details of your protocols)?