E. coli and SDS-PAGE
mm37301 at secc.ecc.u-tokyo.ac.jp
Fri May 20 05:39:54 EST 1994
In article <bushnt.1119742558A at usenet.rpi.edu> bushnt at rpi.edu (Timothy Bushnell) writes:
> In an undergraduate lab, we are having the students prepare protein
>profiles of a stationary phase E. coli culture using SDS-PAGE. Currently,
>we spin 50 mls of culture down and resuspend the pellet in 1 ml of 62.5 mm
>Tris pH 6.8, 2% SDS, 5% B-mercapt. and 10% glycerol. This is then boiled
>for 5 to 10 minutes and the samples then applied to a SDS-PAGE gel.
> The problem is that the sample is very viscous and near impossible to
>successfully load on a gel. Typically, after the sample is loaded into the
>wells, and the pipet tip removed, the sample sticks to the tip and is
>removed as a gob into the upper tank buffer. Very rarely, when a sample is
>overboiled (20+ minutes) and loaded hot, we avoid this problem.
You can easily loer the viscosity by passing the sample through 25-27G
needles for 5-10 times, which shears the E. coli genome.
Hope this helps.
Yasushi Okada, MD.
($@2,ED9/;V(J <= Japanese Kanji)
Department of Anatomy & Cell Biology
University of Tokyo
($@El5~Bg0e!&Bh#12rK6(J <= Japanese Kanji)
Email:mm37301 at secc.ecc.u-tokyo.ac.jp
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