electrophoresis 2d

Mitchell Isaacs misaacs at spam_me_not.student.usyd.edu.au
Mon Jun 25 11:17:26 EST 2001


"Martin Benoît" <benoit.martin at bordet.be> wrote in message
news:DF1E44F5DBD2D211B2100060087E02B13F6AFB at mail.bordet.be...
>
> Why is it not possible with silver staining ? It seems to me that the
> sensivity is better with the silver staining than with the coomasie
> staining.
Silver staining is certainly more sensitive than Coomassie. However, the
linear range of silver is very small (ie the range of concentrations of
protein where the intensity of stain is proportional to the concentration is
only very small). In other words, for almost all concentrations of protein,
the intensity of silver stain is not proportional to the concentration of
protein.

Coomassie has a large linear range. Sypro Ruby is not only as sensitive as
silver, but it also has a huge linear range (which encompasses that of both
silver and coomassie).

There are a range of SYPRO dyes - fluorescent stains. Have a look at:

Lopez MF, Berggren K, Chernokalskaya E, Lazarev A, Robinson M, Patton WF.
A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect
to protein detection in two-dimensional gels and identification by peptide
mass profiling.
Electrophoresis. 2000 Nov;21(17):3673-83

Berggren K, Chernokalskaya E, Steinberg TH, Kemper C, Lopez MF, Diwu Z,
Haugland RP, Patton WF.
Background-free, high sensitivity staining of proteins in one- and
two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a
luminescent ruthenium complex.
Electrophoresis. 2000 Jul;21(12):2509-21.

Yan JX, Harry RA, Spibey C, Dunn MJ.
Postelectrophoretic staining of proteins separated by two-dimensional gel
electrophoresis using SYPRO dyes.
Electrophoresis. 2000 Nov;21(17):3657-65.


>
> Which method do you purpose to quantify the protein concentration with the
> coomasie staining ?
With coomassie, the intensity, or optical density of the stain is
proportional to the concentration of protein, AT EQUILIBRIUM. Ie you will
probably need to stain for a few days to ensure you have reached
equilibrium. I'm not sure all types of coomassie are quantitative, the
literature should be available to tell you which are best. Try G-250.

Cheers, and happy staining,
Mitchell





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