Westerns; napthol reagent?

engelbert_buxbaum4 at my-deja.com engelbert_buxbaum4 at my-deja.com
Sat Nov 11 01:19:01 EST 2000

In article <8uc0rn$rkd$1 at nnrp1.deja.com>,
> I have been trying to do westerns using ECL Plus reagent from Amersham
> without success and now I would like to try something that I had done
> using a napthol reagen successfully a long time ago;

You may wish to try nickel-amplified diaminobenzidine instead, which is
more sensitive. The other point to keep in mind (with both ECL and DAB)
is that you get better signal-to-noise with PVDF rather than nylon- or
nitrocellulose membranes. Dunn's Buffer (Anal. Biochem. 157 (1986)
144-53, 10 mM NaHCO3, 3 mM Na2CO3, 20% MeOH, pH 9.9) is cheaper than
Towbin's for blotting and gives higher sensitivity. Since I am working
with large membrane proteins, I substitute 100 uM SDS for the MeOH.

For nickel-intensified DAB staining blot your gels, block and develope
with primary and HRP-labeled secondary antibody as usual. Keep in mind
that HRP is poisoned by azide, so this preservative must not be used.

For the colour reaction you need:

20-times buffer: 6.01 g Tris, 14.6 g NaCl, 0.95 g NiCl2*6 H2O in 40 ml.
Adjust to pH 7.6 and add 250 mg DAB. Make to a total volume of 50 ml,
filter and freeze in 1 ml aliquots.

Note that DAB is a suspected carcinogen, and needs to be handled with
reasonable care.

1 ml of the concentrate and 19 ml water mixed with 10 ul 30% H2O2 give
the staining solution. Stain at room temperature until your bands appear
black on a white background (on PVDF 30 min to over night, depending on
the required sensitivity. On nitrocellulose, strong background appears
after about 2 h).

This method may also be used for staining in immuno-histology, after
labeling with peroxydase-antiperoxidase complex.

In E. Kessler (ed): Nonradioactive Labeling and Detection of
Biomolecules, Berlin (Springer) 1992 a gold amplification for blots
developed as above is described, but in my hands that increases the
background more than the signal.

The bands are stable for years, they do not bleach as with some other
staining methods.

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