I am doing western blot to detect the activation of Rac1 (Rac1-GTP). VAV2 is known to activate Rac1. I want to confirm by western blot.
The length of Flag-C1 (control) is shorter than Flag-VAV2 (size of VAV2: 2637 nu). Should I use more DNA of VAV2 when I transfect DNA to the mamalian cells? I want to equal the protein expression of these 2 constructs in the cells.
For example: if I use 2.5 ug of Flaf-C1 DNA, I will use 6.25 ug of Flag-VAV2.
Thanks
The size of your insert is not the only aspect you need to consider in order to achieve equal amounts, some DNA sequences are better overexpressed than others maybe because of the different DNA secondary structure. I recommend you titrate your plasmids in a small scale experiment and check the result by western blot. You can use a 24well plate and combine different amounts of each DNA (i.e 1:1, 1:2, 2:1, 1:3, 3:1, etc). But remember to maintain the same amount of total DNA since this quantity is linked to total cell number.
- Badges
- Science method