"Artem Evdokimov" <AEVDOKIMOZ at cinci.rr.com> wrote:
>Are you sure that a 'homebrew' silver stain isn't good enough for what
>you're doing ?
Yeah... I am, in fact, quite sure that most of my homerewed batches
work better than most commercial kits.
>For example, here's an approximate protocol that we have developed cause we
>hate waiting for silver stain. It's quick-n-dirty (that is it gives slightly
>higher background and slightly less sensitivity than full-blown 6-hour
>protocol) but it works well enough for most of our purposes.
>>1) fix gel (microwave in your favorite AcOH/MeOH/H2O mixture for 1 min on
>high)
>2) Wash gel (microwave 1 minute 3 times in three changes of H2O)
>3) <1 min microwave in 0.1% AgNO3 (heat to at least 70C, but do NOT let it
>boil!)
>4) Wash gel (microwave 1 minute 2 times in two changes of H2O)
>5) <1 min microwave in 0.2% Na2S2O3 (heat, but do NOT let it boil!)
>6) Wash gel (microwave 1 minute 2 times in two changes of H2O)
>7) Develop gel with standard carbonate-HCHO mixture (look at bands
>appearing, stop when ready with excess of AcOH/MeOH/H2O mixture). Generally
>under 2 minutes.
>8) store in AcOH/MeOH/H2O mixture or if needed, gently destain with acidic
>thiosulphate.
>>Total time needed - well under 30 minutes.
I use a different protocol (used to buy Bio-Rad's kit lomng ago, then
looked up the reference and made it myself; it's under 45 min and no
microwave).
DK
