Tryptophan from PAGE gel hydrosylate

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5 total messages Started by Tony Larson Tue, 05 Jun 2001 16:33

#2266

Author: Tony Larson
Date: Tue, 05 Jun 2001 16:33

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I'm looking for advice on the best way to hydrolyze a protein band cut out
from a PAGE gel to its component amino acids, and then to derivatize and 
analyze these amino acids by HPLC or GCMS.  One of my main concerns is that
that tryptophan and hydroxyproline are major constituents of the protein in question,
so I don't want to lose these in the hydrolysis or derivatization procedures.
My initial thought is that I would need to use methanesulfonic acid hydrolysis
(rather than HCl) to preserve the tryptophan, but as MSA is not volatile, I am
unsure how I should treat the hydrosylate for derivatization.  To summarize, I'd
like to know:

1) Are there any special procedures needed for hydrolysis of excised PAGE gel slices?
(especially with MSA as opposed to HCl)

2) Are there background contamination issues with gel hydrosylates?

3) Is there an amino acid derivatizing procedure compatible with residual MSA?
(I've used OPA, PTC, and DABS-Cl methods for HPLC and TMS derivatives
for GCMS of dried amino acids).

4) Is there a procedure for partitioning amino acids away from MSA hydrolyzed
protein?

thanks
Tony

Re: Tryptophan from PAGE gel hydrosylate

#2261

Author: jose
Date: Wed, 06 Jun 2001 18:37

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i have, in the past, used the following basic recipe to get protein
bands out of PAGE gels for HPLC (and later MS) analysis:

1. excise the band after sample elucidation (ie autorad, staining)
2. wash the slices 2x5 min in 50% methanol
3. wash in 1x5 min with ddH2O
4. add 0.5ml ddH2O and place on a rocker overnight at 4'C
5. spin samples at 14k, 10 minutes in microfuge
6. remove supernatant and spin this down again for 10 min at 14k
7. dry this supernatant down
8. resuspend in 100ul 50% formic acid, redry

hope this helps. 

another procedure i used was (again in an eppendorf, prep for HPC
analysis):

1. take gel slices and add 1ml 50% MeOH, rocker at RT for 1h
2. wash with HPLC water for 30 min
3. wash twice with 1ml 50 mM Ammonium Bicarb (in HPLC water) 5 min
4. add 0.5 ml 50 mM Ammonium Bicarb (in HPLC water), 37'C
5. add 20 ul 1mg/ml trypsin, incubate for 4h at 37'C. repeat four more
times
6. remove gel band, dry down sample (speed vac), keep at -20'C until use

if you didn't excise enough protein, repeat (the gel bands can be kept
at -20'C for a while). 

hope this helps,

jose nazario				jose@cwru.edu

Re: Tryptophan from PAGE gel hydrosylate

#2262

Author: ckf
Date: Mon, 11 Jun 2001 17:27

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jose

I read your method for trypsin digests of PAGE samples. Is the method (and
subsequent reverse phase HPLC) compatible with SDS from denaturing gels or
is the method only compatible with native PAGE

cheers

Craig

Re: Tryptophan from PAGE gel hydrosylate

#2263

Author: jose
Date: Mon, 11 Jun 2001 21:05

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ckf wrote:

> I read your method for trypsin digests of PAGE samples. Is the method (and
> subsequent reverse phase HPLC) compatible with SDS from denaturing gels or
> is the method only compatible with native PAGE

i think i use it with SDS containing PAGE gels, too. it may be worth
your while to investigate the use of a heartier protease, one that is
SDS resistant, if you suspect thats a problem.

jose nazario				jose@cwru.edu

Re: Tryptophan from PAGE gel hydrosylate

#2264

Author: Mitchell Isaacs
Date: Tue, 12 Jun 2001 02:00

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"ckf" <craig@fulton52.freeserve.co.uk> wrote in message
news:9g2vaf$i26$1@news5.svr.pol.co.uk...
> jose
>
> I read your method for trypsin digests of PAGE samples. Is the method (and
> subsequent reverse phase HPLC) compatible with SDS from denaturing gels or
> is the method only compatible with native PAGE
If you check out the technical bulletin for promega trypsin,
(http://www.promega.com/tbs/tb512/TB512.pdf) you will see that it is
resistant to mild denaturing conditions, eg 0.1% SDS.

Cheers,
Mitchell

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