In article <4b6nq4$a0p at gazette.bcm.tmc.edu>, ah690549 at bcm.tmc.edu (Annette
C. Hollmann) wrote:
> Are there any decent prestained molecular weight markers for SDS-PAGE
> available? Right now I am using markers from Sigma, and the bands are
> always 1/4 inch wide and very faint. My samples run ok, so the problem
> is definitely the markers and not the gel. I have to use prestained
> markers because I'm using the gels for Western blots. If I were staining
> the gels I'd just use a mix of purified proteins for a "homemade"
> set of markers. I need the size range of 20 to 90 kDa, which should
> be fairly common. I know I could just leaf through some catalogs
> and order a few batches of markers to test, but I don't want to
> risk wasting money on markers that are just as low quality as the
> Sigma stuff.
I had the same experience with several brands of prestained marker
(rainbow or blue stained markers from Amersham, BioRad, GIBCO etc...).
They never give sharp bands, even if you use a Fling and Gregerson Gel
which produces much sharper bands than Laemmli (Ref. is Anal Biochem 155:
I used my normal Pharmacia markers (LMW, MW-range 14.4-94 kDa, 6
Marker-bands) which gave me the best results and stained the NC membrane
with fast green as follows
1) Incubate the blot 30 sec to 5 min (30 sec is usually sufficient and
will shorten your destaining time) in a solution of 0.1% Fast Green FCF
(e.g. Sigma F-7252) in 1% Acetic Acid while gentle shaking.
2) Wash away background with destilled water (several water changes if
necessary; if you don't want a photograph a copy on a standard xerox
machine often works for your lab jounal, I usually marked the position of
the makers on the blot with a pen or with a needle)
3) Destain about 2-10 min with 200 mM NaOH, change the NaOH at least twice
(If your protein concentration was very high and you stained too long some
of the dye might remain where strong bands are present-this color will
dissappear when incubating with the blocking reagent).
4) Wash twice with TBS before blocking the membrane.
The procedure takes usually not more than 5-10 min, the dye is a food dye
and thus nontoxic and the overall procedure is extremely cheap. You can
reuse the Fast Green solution as many times as you want (or until your
bottle with the stock soln. is empty). Important: This procedure does not
work with PVDF membrane (the dye will stick irreversible to the PVDF)- you
can stain PVDF reversivble with Poanceau red (as already posted,
disadvantage is that the sensitivity with the Poanceau red stain is
slightly lower). Another advantage is that the procedure requires no
organic solvents which makes your membrane shrink.
Give it a try
>ah690549 at mbcr.bcm.tmc.edu
Sender: Thomas Urbig
Institute for Molecular and Cellular Biology
16 Divinity Ave.
Cambridge, MA 02138
Tel: (USA) 617 495 3716; Fax: (USA) 617 495 9300
Email: Thomas at hastingslab.harvard.edu