Where is the protein ladder?

[anacss]

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...

Thanks!
Ana Carolina

[danfive]

90 min seems a long time. You may be blowing the marker and proteins through the blot paper.
Your gel percentages are not atypical, and should not cause any problems for the transfer.
Bottom line manipulate the voltage and time of transfer (keep amps constant).
Also you can stain the gel and blot to test transfer efficiency.

[danfive]

As to the sample not entering the 8% gel, I would recommend repeating, could just be a random human error during sample prep or gel setup.
And remember if you are interested in large proteins they will take more time to transfer than small proteins (so time is a factor in this case).