SDS-PAGE help badly needed

Giorgio Spagnol spagnol at galactica.it
Wed Jul 26 11:02:55 EST 1995


Dear netters,

this is really becoming a nightmare! Since two weeks protein samples
refuse to enter my one dimensional SDS-PAGE minigel.
Funny thing: only the MW markers enter, as normal.
Gel is 6% acrylamide, 0.1% SDS, and proteins are of a MW which should
enter the gel (e.g. 67 kD). I think something is wrong in the loading
buffer (salts should not interfere, since in the samples concentrations
are kept in 150 mM or even in H2O  ), but damn if I know what.
This is the composition of the loading buffer: Tris Cl Ph 6.8,
Glycerol, 20% dTT O.1M, Blue Bromophenol 0.2%, fial conc. of SDS: 1%. MW
are in 50% glycerol, 300 mM NaCl, 3mM NaN3, dTT 0.1 M, and I add the
loading buffer to them as to my protein samples, to keep SDS
concentration to 1%. Probably all this information is superfluos, since,
the buffers are the  exactly the same I used before the problem started.
Please help, or I shall start to believe in black magic, and this is not
a point of honour for a researcher.
Thanks!
Giorgio.



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