Hi, I'm an undergraduate student working on western blot.

I have understood most of the principles of western blot but still have some questions about it.

1) I wonder why the transfer time is longer than the loading time. Since the gel's vertical length is longer than the width of it, I think the transfer time should be much shorter than the loading time because the proteins just have to go through the gel and stick to the membrane. Does longer transfer time makes the protein more well-embedded to the membrane?

2) Transfer time difference makes me crazy. In my lab, we transfer in constant voltage of 60v for about 2hours or more. However, I found that some labs run transfer in constant current. And some labs even have a equation like 1.5mA/cm2. What could be different between constant current or voltage? And also, in Bio-Rad Powerpac Basic, I can fix either current or voltage so the other one differs continuously. Since V=IR I can understand the fluctuation of either voltage or current but what is the R in transfer? Is it resistance in the transfer tank, like gel to buffer to membrane, or is the resistance of the device itself?

And also, if the lab uses the equation like 1.5mA/cm2, is it effective to all percentage of gels? I think proteins larger than 100kDa in 15%gel needs more transfer time than lower percentage gels, but the equation has no explanation of the percentage.