Trouble-shooting Tris-Tricine SDS-PAGE - (Dec/11/2008 )
Hello,
I am trying to run a 10-20% pre-cast Tris-Tricine gel from BioRad in search of a ~4kD protein. I am running the gel using seperate Anode and Cathode buffers (see recipes below). I have tried various running times and conditions, so far the best has been 40V/100mA for ~7 hours. I feel this is way too long but it has worked the best so far, I have also tried running @ 30V for 1hour then turning up to 100V for 2 hours but the bands were very fuzzy and the dye front had a big smear associated with it.
Anode Buffer:
24.22g Tris
in 1L H2O
pH = 8.9
Cathode Buffer:
12.11g Tris
17.92g Tricine
1g SDS
in 1L H2O
I am heating my samples to 70degrees prior to loading, and I am loading all the wells with an equal volume and concentration of sample. During the first hour of running the dye front is nice and crisp, however after that time the dye front begins to smear in the sample lanes and a half-smile (one side of the gel only) begins to form. The smearing only gets worse the longer the gel runs and sometimes it is so bad it overlaps the area where I would expect to find my protein of interest. Does anyone have any ideas as to what could be causing this smearing and half-smiling? Any suggestions would be helpful. Thank you!
how far do you run your dye front?
i have seen where the dye spreads then contracts near the end of the gel. you can see a second buffer front at the back of the dye smear (you can see convection). we let the front reach the end, the dye sharpens and so do the bands. the gel does not overrun.
the smear could also be caused by improperly prepared buffer. check the pH of the buffers and make sure that they are correct (don't adjust the cathode buffer, if it is off then make it again, carefully, also remember that sds will interfere with pH reading). it can also be caused by old sds.
a one-sided smile can be caused by a dirty electrode wire. check that the wire is clear for its entire exposed length.
yes, be careful with SDS. I also had smear on tris tricine gels that was solved by changing the SDS (we were buying SDS solution that was precipitating, I replaced it by preparing my own solution with an old stock of powder SDS. no precipitation. no more problem wtih tricine gels)
Do you cool your system while running ?
i have seen where the dye spreads then contracts near the end of the gel. you can see a second buffer front at the back of the dye smear (you can see convection). we let the front reach the end, the dye sharpens and so do the bands. the gel does not overrun.
the smear could also be caused by improperly prepared buffer. check the pH of the buffers and make sure that they are correct (don't adjust the cathode buffer, if it is off then make it again, carefully, also remember that sds will interfere with pH reading). it can also be caused by old sds.
a one-sided smile can be caused by a dirty electrode wire. check that the wire is clear for its entire exposed length.
I have let the dye front come relatively close to the edge of the plate, I will try to run it a little longer next time to see if the smear pulls itself together, I am just afraid of running my protein off because it is only 4kD. I am preparing my buffers fresh the day that I use them and I have checked the pH following the complete prep and it seems to be fine. I just purchased my SDS last month so I don't think that could be the source of my problem. I will try cleaning my electrode wire more thoroughly to get rid of the half-smile. Thank you for all of you thoughts and suggestions, I will try a few more things to see if I can resolve this!
Do you cool your system while running ?
I have not yet tried cooling the system while I run. I will try putting it in the cold room next time to see if this helps. Thanks for the suggestion!
another thing to consider is the medium that the sample is in before adding sds sample buffer. the salts and buffers can affect the appearance of the sample when it is run.