Hello,
I've been having trouble recently with my western blot experiments on cell conditioned media. I'm using a home-made 10% SDS gel, as well as a home-made migration buffer and loading buffer.
When migrating my conditioned media, half way through the migration process, when the migration front is in the middle of the gel, this same migration front starts to form a curve where the middle samples seem to run more slowly than the samples at both ends.
Some additional observations:
- I do not get this problem with classic protein samples, only conditioned media
- I don't think it's a gel problem, as I produce my protein and conditioned media gels at the same time
-I don't think it's a problem of uneven electric field as protein samples running in the same gel holders/same migration tank do not have this problem.
Could someone shed some light on this conditioned media mystery ?
Thanks,
Thomas
Dear Thomas,
Smiling within a lane and across lanes in a gel is often the result of different salt levels. You could try dialysing each of your samples for 10 - 30 minutes using a common buffer (e.g., TE for DNA, lysis or final suspension buffer for proteins) before adding the SDS-PAGE loading buffer, denaturation and loading.
Andrew
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