Dear All,
I wanted to post an Idea, Method for detection of oxidized proteins in 2D gels.
I started using the Oxyblot kit from Chemicon.
I will post the protocol later on this week.
The Idea is to incubate the IPG strip after the isoelectric focusing with 2,4-dintrophenylhidrazine (DNPH) for 15 minute wash it and then run the second dimension SDS-PAGE. Then doing western and using an Anti DNP Ab.
Guy

Hi,
This is The Prtocol from Chemicon web page:
www.chemicon.com/Resource/ litlibrary/924OxyBlotflyer.pd

Assay Protocol:
1. Prepare protein lysate from your cells using
any of a variety of protocols.
2. Derivatize your protein sample:
a. Aliquot 15-20 ?g of protein in 5 ?L into 2 tubes.
b. Denature the protein by adding 5 ?L of 12% SDS.
c. Add 10 ?L of 1X DNPH to one set of tubes and
10 ?L 1X Derivatization-Control Solution to the
other set.
d. Incubate for 15 min @ RT.
e. Add 7.5 ?L Neutralization Solution to each tube.
3. Run the samples on an SDS-PAGE gel.
4. Transfer the gel to nitrocellulose or PVDF membrane
and incubate 1 hr in Blocking/Dilution buffer.
5. Dilute the 1° antibody 1:150 in Blocking/Dilution
buffer just before use. Incubate the blot for ~1 hr at
18° to 25°C with gentle shaking.
6. Rinse the membrane 3X with 1X PBS-T.
7. Dilute the 2° antibody 1:300 in Blocking/Dilution
buffer and incubate the blot for ~1 hr at 18° to
25°C with gentle shaking.
8. Rinse the membrane 3X with 1X PBS-T.
9. Drain the excess buffer and treat with
chemiluminescent reagents.
10. Expose to film.

The changes that I have used in order to do it with my 2D sample are as follows:
The Samples are applyed to the 2D IEF strip than rehydrated and Focused.
The strips are then incubated with the 2m 1 X DNPH in 15ml tubes for 15min rocking at room temperature.
The stips are washed with 2MHCl in ddH2O.
The strips then are ready for regular equlibration and 2nd D SDS-PAGE.

Transfer to membrane and prob with anti DNPH
Guy