Hi,

I am preparing to do a western blot and I need to add 4X Laemli sample buffer to my samples before adding them to the gel and I had a few questions (I am using the Biorad 4X Laemli sample buffer):

1. If I want to prepare 250 uL of 4X Laemli sample buffer, then should I just add 25 uL of a reducing agent (10X DTT in my case) to 225 uL of 4X Laemli sample buffer to make the reducing agent 1X?

2. Assuming that I was correct in my previous question, if I have 15 uL of protein sample then I should add 5 uL of 4X Laemli sample buffer to make it 1X right?

3. Also, why do we add a reducing agent to the sample buffer? Is it to reduce the disulfide bonds of the proteins so that they have a more linear structure and separate better in the gel?

Thanks for the help! I really appreciate it.