Free Sulphydryl detection

David Martin damartin at bioslave.uio.no
Thu Mar 21 03:26:14 EST 1996


In article <Pine.SUN.3.91.960320114347.21539A-100000 at chuma>,
   leverone at CHUMA.CAS.USF.EDU ("Marianne Leverone ", BIO) wrote:
-->Record Absorbance at 412 nm.  DTNB Coefficient is 13,700 M-1, cm-1.
-->
-->Reference: Fong and Schulz.  1981.  Methods in Enzymology.  71:390-398.
Actually the original reference is in 1959, and DTNB is named after the 
co-author as Ellmans(?) reagent (I can't remember the name properly)
I used it and it gives good results.

-->On Tue, 19 Mar 1996, Achim Recktenwald wrote:
-->
-->> Michael Daws wrote:
-->> > 
-->> > I have a 19-mer peptide (2200 MW) with a C-terminal cysteine which I
-->> > disulphide bonded.  Is there any nice easy colorimetric reagent I can
-->> > use to detect free sulphydryl groups so that I can determine just how
-->> > much of my peptide is disulphide bonded?  Alternatively does anybody
Try Absorbance at 250 nm

-->> 
-->> A gel is easy. Just run a SDS-gel, but run two samples.Treat one sample 
with 
-->> reducing SDS-buffer, that means it contains mercaptoethanol or DTT, the 
other 
-->> sample with a SDS-buffer without reducing agent.
To resolve these peptides you will need to use a tris-tricine gel (see 
Schagger and von Jagow (1987) Analytical Biochemistry 166, 368-379)

Conventional tris-glycine will not give you the resolution you require.

-->> If you are correct, the reduced sample should show only one band at 2200 
Da, but 
-->> the non-reduced sample a major band at 4400 Da, and a minor one at 2200 
Da.
-->> 
-->> You'll probably have to run a gradient gel or a highly crosslinked one. 
about 15% T should be fine.

Good luck!

..d

* David Martin, PhD -  Post-Doctoral Research Fellow    *
* Atherosclerosis and Thrombosis research group         *
* Biotechnologisenteret i Oslo                          *
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