Nile red.

Paul N Hengen pnh at fcsparc6.ncifcrf.gov
Wed Jun 8 18:01:47 EST 1994


 In article <1994Jun8.192936.171297 at eros.embl-heidelberg.de>
 houthaeve at embl-heidelberg.de (Tony Houthaeve) writes:

> I'm thinking of detecting proteins using a dye such as Nile Red or SYBR Red
> from Molecular Probes and then slicing the protein band directly from the gel.
> Is it possible to sequence a protein directly from a polyacryamide gel without
> blotting onto PVDF? Do you foresee any problems with the red dye blocking the
> microsequencing? What is the minimum amount of protein needed for sequencing?

| It is not possible to sequence a protein within a polyacrylamide gel, so you
| first have to elute the protein out of their or blot it onto PVDF (nitrocellulo
| se does not work).  Nile red is a good stain, it has hydrophobic properties and
| in only a few minutes a high fluorescence in protein bands in gels is seen.
| More it is possible to first stain your gel with Nile red and then additionally
| blot onto PVDF. Subsequent sequencing can then be performed, Nile red does not
| block.  see : Biotechniques vol 16, No 4 (1994) p 621-624 An article from
| Bermudez, A, et al.

Yes. That's where I got the idea from, but it doesn't make sense to me. In
that paper, the authors first stain with Nile Red, then electrotransfer to
PVDF, then stain with Coomassie Blue before removing the protein for micro-
sequencing.  They further say that individual bands could be excised and
electroblotted onto PVDF in a mini-sandwich. Since the level of detection with
Nile Red is a banded protein of about 20 pg, I don't see why you can't just
remove the band directly from the gel for sequencing. I'm looking for the best
way to recover the band for sequence analysis without transfer to PVDF. Which
should I use? electroelution? freeze and squeeze? Centricon? Which works best
for maximum yield of protein and sequence data? Or is PVDF the only way to fly?

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh at ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
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