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.
Needless to say, the undergraduates are unsatified with the results.
Help.
If anyone has a better idea on preparing the samples in a short amount
of time (under an hour) please let me know. Several ideas that we have had
include acetone precipitation, lysozyme treatment and DNase treatment.
Thank you in advance for any assistance.
Tim Bushnell
Rensselaer Polytechnic Institute
Timothy Bushnell
Rensselaer Polytechnic Institute
Plant Research Group
These in days when heavens mouring/ the days when earths foundation shook/
followed their mercenary calling and took their wages and are dead.
Their shoulder held the sky suspended/ they stood and earths foundation
stayed/ What god abanded, these defended/ And saved the sum of things for pay
