SDS-PAGE help badly needed

c635499 at black.missouri.edu c635499 at black.missouri.edu
Tue Aug 8 19:06:30 EST 1995


   Did you wash the wells with running buffer before loading?  Unpolymerized
gel solution prevents the sinking of samples in the well, unless the glycerol
conc. is very high, like in the MW marker .

Nanfei
  
f.r.byrne (gen126 at abdn.ac.uk) wrote:

: sounds like you are doing everything right.
: there's always that old reliab;le that
: someone borrowed them and neglected
: to ask your permission.
: make 'em up again yourself and see.
: what more can I say?

: Fergus
: University of Aberdeen


: Giorgio Spagnol (spagnol at galactica.it) wrote:
: : 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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