5 kDa protein detection

Achim Recktenwald achimr at home.com
Tue Jan 5 00:39:36 EST 1999


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
>





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