5 replies to this topic

[Trying-2-Graduate]

I have a cell lysate in which I perform protein precipitation on. I then resolubilize my proteins in Laemmli buffer and run on an SDS-PAGE. I visualize the proteins via Coomassie staining. My problem is that my protein bands are darker in the center than they are on the sides. I have attached a pic to explain what I mean.
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I am using gels purchased from BioRad, and fresh running and laemmli buffer. This has happened more than once, so I doubt it is an issue with the gel itself.

Any ideas on whats going on and how to fix it?

[bob1]

What shape are your wells? They look square, but brighter in the middle often means V shaped wells.

Edited by bob1, 14 September 2009 - 05:37 PM.

[HomeBrew]

Are you careful to only go part way into the well with the tip when loading, allowing the sample to sink to the bottom of the well when discharged? I wonder if burying the tip into the bottom of the well could produce this type of artifact?

[fysio lab]

Maybe too much salt in your samples?
grs

[Trying-2-Graduate]

My wells are square, not V-shaped.

During loading, I have been careful to not push into the well. I have the tip in the middle and load, letting the sample sink to the bottom.

If there is salt in my samples, I have no clue where it is coming from. I do a protein precipitation before using Calbiochems proteoextract kit, then resolubilize the proteins in loading buffer. According to calbiochem, there should not be anything in the protein pellet wash solution that would interfere with running the a 1D SDS-PAGE (in case my pellet is not totally dry before resolubilizing it)

[fysio lab]

sja..the loading buffer is a commercial one or self-made? (maybe for adjusting the pH you added salts?) . Other thing: your sample isn't viscous?