Please someone can help me?
I have a lot of problems with western blotting but i need to study the expression/activation of various proteins.
First, yesterday the protein ladder (Fermentas) disappeared during the transfer step in my Western blotting but the conditions were the same I ever used, 400 mA (constant) for 90 min. Such event already happened last week although after the first event I had not problem until yesterday.
Second, although i used to double the time of transfer when using 1.5 mm gel i not certain about this. If i tranfer proteins from a gel of 0.75 mm in 45min/400mA so the transfer from a gel of 1.5 mm must be done in the double time (90 min)?
Third, changes in acrylamide concentration also influences the time of transfer?
Forth, i tried to work with a gel of 8% acrylamide but for my surprise de sample could not enter the gel during the running! (100V) At the same time i do my tradittional 12% gel and the running of it do with no problems! What is the problem?
Sorry for too much questions...