Re: Misc. - Pre SDS PAGE reduction and alkylation

Margaret Schott (SCHOTTM@gunet.georgetown.edu)
Mon, 29 Jun 1998 11:50:59 -0500

Just a few comments on reduction / alkylation prior to running
SDS-PAGE. There is a scenario pertaining to antibody proteins,
which have two sets of disulfide bonds (at least when speaking
of accessibility to chemical manipulations). One type are the
intrachain disulfide bonds, which are buried and for all practical
purposes inaccessible to solvents and reagents under neutral
aqueous (physiological) conditions. The other type are the
interchain disulfide bonds, which are readily cleaved by reducing
agents and readily modifiable by reagents.

Thus one can selectively reduce and alkylate the interchain
disulfides prior to running a gel. Typical conditions (described in
my paper from Bioconjugate Chemistry, March/April issue, 1993)
would include reduction with 10 mM DTT at R.T. for 15-30 min
in phosphate buffer (0.1 M, for good buffering capacity; pH 7.0
or 7.2), followed by alkylation with iodoacetamide (reacts faster
than iodoacetic acid although excess of either reagent should do
the job) at 50 mM (to give >2-fold excess over DTT thiols) for 15-
30 minutes at R.T. After that you can mix the samples with 2x
sample buffer for gel running. One advantage of this specialized
situation for antibodies is that the different chains can be observed
under non-reducing conditions. Perhaps this method could apply
to other proteins with both buried and solvent-accessible disulfides,
allowing a unique look at gel band patterns.

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Margaret E. (Peggy) Schott
Synthesis & Sequencing Shared Resource
E401 Research Bldg, Georgetown University
3970 Reservoir Rd, NW, Washington, DC 20007
(202) 687-1684 FAX (202) 687-7505
Email: Schottm@gunet.georgetown.edu
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

>>> Ken Mitchelhill <k.mitchelhill@medicine.unimelb.edu.au> 06/22 7:12 PM >>>
Dear ABRF list,

In the past, I have always reduced my sample prior to running on SDS PAGE
and followed the standard protocols for reduction/alkylation of gel slices
to obtain alkylated cysteines if required. I now have the need to alkylate
just prior to SDS PAGE.

Would those out there who routinely reduce and alkylate their samples PRIOR
to SDS PAGE care to share their protocols? I have tried to glean a few
protocols from the literature and have tried the following:

Sample in standard SDS sample buffer (60mM Tris pH 6.8, 2% SDS)
made 10mM DTT by addition of 1M DTT (in water)
100 degrees celcius for 10 minutes
cooled to room temperature
made 50mM iodoacetamide by addition of 1M iodoacetamide (in water)
room temperature in dark for 15 minutes
made 2% mercaptoethanol to quench iodoacetamide by addition of neat
mercaptoethanol.

I notice the bromphenol blue in the sample buffer showing a mild drop in pH
during the alkylation step and it goes quite yellow some time after the
addition of the mercaptoethanol and needs quite a few microlitres of
concentrated Tris to get the pH back to normal running levels.

Is this pH change normal?

Regards....Ken

********************************

Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA

Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676

Email: k.mitchelhill@medicine.unimelb.edu.au

Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.abrf.org

***********************************

>>> Ken Mitchelhill <k.mitchelhill@medicine.unimelb.edu.au> 06/22 7:12 PM >>>
Dear ABRF list,

In the past, I have always reduced my sample prior to running on SDS PAGE
and followed the standard protocols for reduction/alkylation of gel slices
to obtain alkylated cysteines if required. I now have the need to alkylate
just prior to SDS PAGE.

Would those out there who routinely reduce and alkylate their samples PRIOR
to SDS PAGE care to share their protocols? I have tried to glean a few
protocols from the literature and have tried the following:

Sample in standard SDS sample buffer (60mM Tris pH 6.8, 2% SDS)
made 10mM DTT by addition of 1M DTT (in water)
100 degrees celcius for 10 minutes
cooled to room temperature
made 50mM iodoacetamide by addition of 1M iodoacetamide (in water)
room temperature in dark for 15 minutes
made 2% mercaptoethanol to quench iodoacetamide by addition of neat
mercaptoethanol.

I notice the bromphenol blue in the sample buffer showing a mild drop in pH
during the alkylation step and it goes quite yellow some time after the
addition of the mercaptoethanol and needs quite a few microlitres of
concentrated Tris to get the pH back to normal running levels.

Is this pH change normal?

Regards....Ken

********************************

Ken I. Mitchelhill
The John Holt Protein Structure Laboratory
St. Vincent's Institute of Medical Research
41 Victoria Parade
Fitzroy 3065 Victoria
AUSTRALIA

Telephone: 61-3-9288 2480
Facsimile: 61-3-9416 2676

Email: k.mitchelhill@medicine.unimelb.edu.au

Laboratory: http://www.medstv.unimelb.edu.au/WWWDOCS/SVIMRdocs/JHPSL.html
ABRF: http://www.abrf.org

***********************************

!
!
!
!
!
!