Overexpressed protein not detected - Oh, the frustration... (Mar/16/2010 )
Hello everyone,
I'm having an issue with a western. I have transformed Arabidopsis to overexpress my favourite gene. Prior to transformation, the construct was sequenced and it resulted to be just fine. I got several lines and extracted total protein from 500 mg of tissue using 2x sample buffer. An SDS-PAGE was performed followed by a western. By the time I did the colour development I saw nothing, several times. It is becoming very frustrating.
Has anybody here extracted total protein from plants for Western blotting? If you have any ideas of what could be going on, please HELP.
-Pancho-
Pancho on Mar 16 2010, 10:09 PM said:
Hello everyone,
I'm having an issue with a western. I have transformed Arabidopsis to overexpress my favourite gene. Prior to transformation, the construct was sequenced and it resulted to be just fine. I got several lines and extracted total protein from 500 mg of tissue using 2x sample buffer. An SDS-PAGE was performed followed by a western. By the time I did the colour development I saw nothing, several times. It is becoming very frustrating.
Has anybody here extracted total protein from plants for Western blotting? If you have any ideas of what could be going on, please HELP.
I'm having an issue with a western. I have transformed Arabidopsis to overexpress my favourite gene. Prior to transformation, the construct was sequenced and it resulted to be just fine. I got several lines and extracted total protein from 500 mg of tissue using 2x sample buffer. An SDS-PAGE was performed followed by a western. By the time I did the colour development I saw nothing, several times. It is becoming very frustrating.
Has anybody here extracted total protein from plants for Western blotting? If you have any ideas of what could be going on, please HELP.
Dear Pancho.. it wud be better if u can provide us with more details so that we can pitch in!!!
details like... what is the molecular weight.. wat are the antibodies... wat conditions u are using for sds and western , what is the detection system?etc!!
-Pradeep Iyer-