Larger protein size in SDS gel - why is it like tat? (Feb/09/2007 )
QUOTE (Aimster @ Sep 21 2007, 11:58 PM)
QUOTE (alice! @ Sep 21 2007, 08:54 AM)
QUOTE (Aimster @ Sep 21 2007, 02:08 AM)
You can add the gel system to your list too - the same protein may have slightly different apparent MWs when you change the gel percentage, gradient, buffers, etc.
but my gel system is same for the marker as well as my protein...?!!!?? plss explain! u mean the mol wt marker bands do not represent their true MWs?? thankuuuu!
Since the amino acid composition and secondary structure of your marker proteins is not the same as your protein of interest, it may be affected differently by changes in the gel system. Also, your markers are typically prepared in a different sample buffer (different conditions with respect to SDS, reducing agent, etc) and may be treated differently before loading (heated or not). The marker proteins usually have been tested thoroughly by the manufacturer to be sure that they run pretty reproducibly, and stay denatured. But your protein of interest is going to be unpredictable. There might be a gel system where you can get this protein to run right where you expect it to be relative to the markers - or there might not.
I've had proteins run a bit high or low many, many times. Generally speaking, it's just something you have to accept - once you've proven to yourself by whatever means are available that it IS the right protein. You could spend months tinkering and never get that protein to run exactly at 22 kDa. Apparent MW by electrophoretic mobility is NOT that same thing as calculated MW, and you can't always expect them to match perfectly. It's a bummer, but unfortunately it's true.
Can I ask, how large is the 6x His tag? Is it 0.84kDa? Because I also have the same problem with expression with pQE30 in M15 cells. My protein is 18kDa but seems to be 23 or 24 kDa. I really don't know why. I suppose what is stated by the people replying could be taken into account. Thank you! heehee but the dillema now is, how do we prove it when you publish your paper that there are such things?
-platyhelminthes-
Hey,
You have to add the mol wt of each of the amino acids between your protein and the tag and also the 6 His . So it will depend on the site where your gene is cloned.
Good Luck
QUOTE (platyhelminthes @ Aug 3 2008, 11:52 AM)
QUOTE (Aimster @ Sep 21 2007, 11:58 PM)
QUOTE (alice! @ Sep 21 2007, 08:54 AM)
QUOTE (Aimster @ Sep 21 2007, 02:08 AM)
You can add the gel system to your list too - the same protein may have slightly different apparent MWs when you change the gel percentage, gradient, buffers, etc.
but my gel system is same for the marker as well as my protein...?!!!?? plss explain! u mean the mol wt marker bands do not represent their true MWs?? thankuuuu!
Since the amino acid composition and secondary structure of your marker proteins is not the same as your protein of interest, it may be affected differently by changes in the gel system. Also, your markers are typically prepared in a different sample buffer (different conditions with respect to SDS, reducing agent, etc) and may be treated differently before loading (heated or not). The marker proteins usually have been tested thoroughly by the manufacturer to be sure that they run pretty reproducibly, and stay denatured. But your protein of interest is going to be unpredictable. There might be a gel system where you can get this protein to run right where you expect it to be relative to the markers - or there might not.
I've had proteins run a bit high or low many, many times. Generally speaking, it's just something you have to accept - once you've proven to yourself by whatever means are available that it IS the right protein. You could spend months tinkering and never get that protein to run exactly at 22 kDa. Apparent MW by electrophoretic mobility is NOT that same thing as calculated MW, and you can't always expect them to match perfectly. It's a bummer, but unfortunately it's true.
Can I ask, how large is the 6x His tag? Is it 0.84kDa? Because I also have the same problem with expression with pQE30 in M15 cells. My protein is 18kDa but seems to be 23 or 24 kDa. I really don't know why. I suppose what is stated by the people replying could be taken into account. Thank you! heehee but the dillema now is, how do we prove it when you publish your paper that there are such things?
-Ritachha-