Hello, I want to know if someone ever did a western blot with an expired mops running buffer with precast gels. I used it for my last gel and the ladder was faint on the gel and obviously on the membrane. Furthermore high molecular weights on the lader weren't not separeted so well, the lowest ones were fine. In the previous gels I didn't have the same problem even if the mops was expired. Why am I having this problem now and not before?
Thanks
I have run westerns with expired MOPS as well and it has never been an issue though I do suggest replacing it. If the ladder was faint on the gel itself, it is probably an issue with your marker or the gel.
Hi Marianna:
I have also run westerns with expired MOPS. My proteins separated well,but had a smiling effect in the center of my gel and my gel was really overheated.
Can you tell us what gel system(types) you used?
In the mean time I will suggest you to replace the buffer(cheap).
Regards,
Rachel
There's nothing much in the MOPS buffer actually to expire, only over the time due to storing conditions the pH may change a bit as a result the current flow may not be ideal. Your problem could be that the pre-cast gel wells were not washed properly resulting in residual storage bufferin the wells which could affect the mobility of the high mol wt proteins. I have used expired MOPS many times without any problem. Also repeated Fridge-thaw of the marker could result in the problem you mentioned with the marker. You could try using less protein and running the gel overnight at low voltage.
Thank you so much guys for all of your suggestions. Two days ago I made a wb with a 4-12% Bolt Bis-Tris gel (not expired) and MOPS buffer ( expired 'cause I have only that one), as usual, and I used a new aliquot of ladder (it was not completely new but I thawed it few times) but I had the same trouble. On the techical sheet of the ladder they suggest to thaw it at RT but I always thawed it on ice. They say also to mix gently but thoroughly but I usually vortex it. Do you think that these are good reason to explain my trouble?
Thanks
My ladder is kept frozen at -24 degrees celsius and thawed at room temperature everytime I want to use it. I even vortex it sometimes and spin it down in centrifuge to get all the liquid to the bottom of tube. My ladder has always come out strongly and clearly on the blots. So, I am not sure whether that is the problem. I suppose the ladder quality could be a possible issue. Maybe repeated freeze-thaw cycles reduced the quality/stability of your particular ladder?
I don't know. Data sheet doesn't give any answer about it but it may be because basically ladder marker is composed by protein. My ladder shoud be kept at -20. But it's not the first time that I used it. Next time I will try a new tube to be sure and I will load more ladder.
Thanks
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