Ponceau S staining problem...
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Fri Dec 1 16:04:02 EST 2000
Neal Robert Melvin <nrmelvin at ucalgary.ca> wrote:
:So what is your Ponceau protocol exactly, Dima??
20X stock solution (good forever; dilute 20X into water):
% by weight:
Ponseau-S - 1.8
TCA - 26.8
Sulfosalicylic acid - 26.8
Make 100-200 ml 1X solution, store at rt and reuse
for as long as it stains (year or so). Take blot wash it
under stream of water briefly (to remove methanol
and SDS if any), pour anough Ponseau to cover the
blot completely, slofh in different directions for 10-15 sec
(absolutely no need to stain for 10-15 min as many
do), pour off the dye, wash the blot with distilled water
until background is completely white. Can dry at this
point or proceed immediately with blocking.
Ponseau-S binds proteins resonably tightly at low
pH (<6.0, distilled water is pH 5.8-6.2), and does not
bind at all at pH>7. Therefore, PBS, TBS, etc, ect will
destain protein bands in a matter of minutes.
:On Thu, 30 Nov 2000, Dima Klenchin wrote:
:> "Kevin A. Morano" <kevin.a.morano at uth.tmc.edu> wrote:
:> :I find that you need to make a very dark Ponceau stain, on the order of
:> :a deep cabernet (2-5%??). It also stains better in 5% acetic acid. I
:> :make the stock and reuse it a bunch. The sensitivity is low, so you
:> :would need at least 5-10 ug total protein on the blot.
:> 5-10 ug? My stock made in TCA/sulfosalicilic acid detects 0.1 ug
:> without any problem. Surely less sensitive than Coomassie
:> but good enough so that in most cases I do not need to run
:> a separate gel for total protein staining. Blot -> stain -> scan ->
:> destain in pH > 7.5 -> block and do Western.
:> - Dima
More information about the Methods