Something I forgot to mention.
You describe your peptide as having few tyrs, no trps; if it also has only
few basic amino acids, it could be one of those proteins that stain very
badly or not at all with Coomassie. You might have to try a silver stain.
Achim
Achim Recktenwald wrote in message ...
>You could run tricine-gels (Schägger et al., Anal Biochem (1987, vol. 166,
>368 - 379) ; they resolve down to <2kDa; I have even seen peptides down to
>1kDa.
>>If you are afraid of loosing your peptide during staining, try a staining
>method used in IEF, where this is always a problem, even for larger
>proteins; e.g.,
>20min in 20% TCA
>30 - 40min 50% methanol, 10% acetic acid
>2x 20min 5% methanol
>40min 10% glutaraldehyde
>2x 10min wash in water or 5% methanol
>then normal Coomassie-staining.
>>The TCA will denature and precipitate your protein, the glutaralehyde will
>then crosslink the protein molecules contained in a protein-band in the
>gel-matrix, making them unleachable.
>If you are using very thin gels, you can easily shorten the incubation
times
>by up to a factor of 2.
>>If you try this with your 15 - 25% PAA gel and still do not find a band, I
>would say your 5kDa might not be part of your sample.
>>Good luck,
>>Achim
>>>>Ryan Werstuik wrote in message <36902084.79F48475 at telerama.com>...
>>I would try using protein markers (with your smallest at 5kDa), running
>>the sample in a center lane, and 1/2 to 2/3 the current time you are using
>>now. I have also heard/read of using a stain that allows the samples to
>>be seen as the gel runs; however, I can't remember the where.
>>>>I hope this helps,
>>Ryan
>>>>Rasmus Wendelbo Nielsen wrote:
>>>>> I am currently trying to purify a protein of 5 kDa, but are having
>>> problems detecting the protein. As it only contains tree tyrosines and
>>> no tryptophans the A280 is very low. I have tried running 18% SDS-PA
>>> gels and a 15 to 25% SDS PA gradient-gels, without luck, the protein
>>> probably diffuses out of the gel during staining.
>>> So I am looking for other (easy) ways to detect low MW proteins. Any
>>> ideas would be appreciated!
>>> I have heard that adding coomassie to the running buffer instead of
>>> post-staining the gels could be a solution. Anyone got experience with
>>> that?
>>> Thanks, Rasmus
>>>>
