SDS-PAGE problems: proteins not running entire legnth - (Aug/01/2007 )
I'm having a repeated problem of having my SDS-PAGE not run well. Even though the dye front is running to the bottom, the proteins remain in the upper third-half of the gel. (An example:
I'm having a repeated problem of having my SDS-PAGE not run well. Even though the dye front is running to the bottom, the proteins remain in the upper third-half of the gel. (An example: here
(oops, submitted before I was ready)
My protein is about 25000 kDa. I'm using a 12% resolving layer with a 4% stacking layer. The buffers are at the right pH, I checked. This has happened with multiple sets of buffers and acrylamide solutions. However, it runs well okay sometimes. (About 20%) I've changed the buffers and acrylamide and still get the problem. Other people in my lab have had the problem as well. (I'm a new student to the lab, but it's not my technique- someone has watched every step that I did for one set, and it still didn't work.)
We're using the Emperor Penguin apparatus from Owl Separation Systems, running the gels at 190V.
Any thoughts on what is going on?
25000 kDa? it is too big! or 25 kDa?, there is a problem even with markers in SDS-Page?
run slower (at 100V).
agree with the previous post? how does the ladder behave tself ?
The protein is 25 kDa, not 25000 kDa.
My concern with running at a lower voltage is that it already takes over an hour to run the gel.
well over an hour seem quick for me
i meant : for running a gel, 1h seems little to me (means too quick).
I would recommend to lower the voltage.
If the whole lab got the same problem, I suspect that there are something wrong with the common stock solutions/equipment. How about asking for either a premade gel or solutions from another lab to see how? Actually, 190V and still over 1h for 12% gel is a bit slow. Did you check the current to see what it is like throughout your run? When you use constant voltage, the change in actual current could indicate if something is wrong with running buffer.
i would do following steps
1-cast a gel with 50% of stacking gel and 50% resolving gel (when you run ur proteins, observe whether they are passing the stacking gel easily, if this is the case then u may have probelm with ur resolving gel)
2- if your proteins remain in upper third part then something to do with ur reagents or power supply.
3- if your power supply is proper then can u check whether u r missing SDS in any reagnets of your electrophoresis?