Streaks appearing between lanes in my western blot - (Oct/05/2005 )

hey all,
i've run a western several times now for PPARg and appearing on my blot I get dark streaks between the lanes that even run into/interfere with my protein imaging.
has anyone experienced this before or know what might be causing it?
i'm attaching an image of a film.
thanks

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looks like contamination from the SB....

is that the way that you label you lanes?

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From my experience you could have 2 different problems (or both):

1. protein purification- your pH is most likely too basic in your prep's, when you add your loading dye, make sure it is blue, if not add (ul at a time) 1M unbuffered Tris to bring the pH back to where the loading dye remains blue (not too much though, this is why you need to add 1 ul at a time)

2. likewise if you are loading small amounts, your sample could be sticking to the well walls, rather than evenly dispersed on the bottom of the well. If you are loading small amounts, fill the well half full with running buffer....and carefully load your sample into the well so that it is now evenly dispersed across the well

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I have found this often happens when your sample has degraded some, or if it is contaminated is some way.

I can't stress enough that having a good sample and preping it well makes the rest of the job so much easier.

Good luck to you!

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