problem with the size of protein - (Jun/05/2006 )
I have been running my Ecoli expressed protein on 12%, 13.5% and 15% SDS page and I always get a double sized expressed protein (around 47kd instead of 23.5kd). I added lots of DTT in my gel loading samples in order to break any possible disulfide bonds but still i am getting a double sized band and not even a tiny amount of right size. what do you guys think about that?thank you for your suggestions
Did you make the expression clone yourself? Have you sequenced your insert? Can you do a western blot to confirm that the band you're seeing is actually your protein?
How are you purifying your over-expressed protein?
Are you using a colored MW marker?
as homebrewed says its important to sequence, you could have cloned two inserts into your vector under certain strategies
I once counter a cytokine that in dimer whenever on SDS-PAGE or other
I do not unstand the reason yet. But the activity of the protein is OK
If the construct is OK, go ahead!
sounds like a dimer to me -
did you cook the sample before loading (5 min 100°C)?