smeared proteins western blot - (Oct/11/2008 )
Hi everyone,
Could someone support me with my problematic western blots.
My protein samples (prepaired from malaria parasites) rather than forming a nice ladder always comming up as a smear (on the coomasie and Pounceau S staining). Additionally my marker is also forming a smear-like lane. I tried 10% poured gels as well as precast once. Please let me know what are yours suggestions how to solve my problem. And by the way I am running my gels at 100V in TG buffer. What is also the expected currence for that conditions for a good buffer?
Very graetful for your support.
Could someone support me with my problematic western blots.
My protein samples (prepaired from malaria parasites) rather than forming a nice ladder always comming up as a smear (on the coomasie and Pounceau S staining). Additionally my marker is also forming a smear-like lane. I tried 10% poured gels as well as precast once. Please let me know what are yours suggestions how to solve my problem. And by the way I am running my gels at 100V in TG buffer. What is also the expected currence for that conditions for a good buffer?
Very graetful for your support.
Your running voltage might be too high. High voltage will also give you "similing" gel. Did you prepare stacking gel? (I know this question is stupid). Your marker shouldn't form smear.
I usually run my protein gel as follows:
25mA during stacking
35mA during separating
All are constant current. I use 10% gel.
Could someone support me with my problematic western blots.
My protein samples (prepaired from malaria parasites) rather than forming a nice ladder always comming up as a smear (on the coomasie and Pounceau S staining). Additionally my marker is also forming a smear-like lane. I tried 10% poured gels as well as precast once. Please let me know what are yours suggestions how to solve my problem. And by the way I am running my gels at 100V in TG buffer. What is also the expected currence for that conditions for a good buffer?
Very graetful for your support.
Your running voltage might be too high. High voltage will also give you "similing" gel. Did you prepare stacking gel? (I know this question is stupid). Your marker shouldn't form smear.
I usually run my protein gel as follows:
25mA during stacking
35mA during separating
All are constant current. I use 10% gel.
Yes I poured the gels myself, but i believe my current was far more nhighier than what you've suggested. I shall try it tomorrow. Thanks
Elkasto
Could someone support me with my problematic western blots.
My protein samples (prepaired from malaria parasites) rather than forming a nice ladder always comming up as a smear (on the coomasie and Pounceau S staining). Additionally my marker is also forming a smear-like lane. I tried 10% poured gels as well as precast once. Please let me know what are yours suggestions how to solve my problem. And by the way I am running my gels at 100V in TG buffer. What is also the expected currence for that conditions for a good buffer?
Very graetful for your support.
Your running voltage might be too high. High voltage will also give you "similing" gel. Did you prepare stacking gel? (I know this question is stupid). Your marker shouldn't form smear.
I usually run my protein gel as follows:
25mA during stacking
35mA during separating
All are constant current. I use 10% gel.
It could also be a problem of your running buffer or gel solutions. Have a look, if you have some fault in your receipts or pH of the solutions of stacking and seperating gel. How about the other solutions? Are they still fresh and good-looking ? 
Do you reuse your running-buffer? Are your plates clean before you cast your gels?
Check you pH. I have seen gels like that when you invert the lower and upper solutions.
Is your SDS in solution? Did you add enough?
Run slower.
Good luck.
A.