OxyBlot Protocol
***OxyBlot Kit Information: OxyBlot Protein Oxidation Detection Kit
S7150
Chemicon International
Store at 4ºC
***NOTE: The amount of DTT (50mM) needed for the Oxyblot procedure is NOT
compatible with any protein assays that we use in the lab. Thus, the each sample
has to be split into 2 tubes (one tube for protein assay and Western blotting, one
tube for Oxyblot).
***Before the assay: For each condition, you will need the following prelabeled
1.5ml Eppendorf tubes:
1) Protein assay
2) Western blot sample using cells that have been lysed with lysis buffer
WITHOUT DTT
3) Sample for Oxyblot (this tube will contain 50mM DTT)
4) Derivatization Reaction for OxyBlot
5) Negative Control for OxyBlot
-----------------------------------------------------------------------------------------------------------Cell harvest has to proceed rapidly to prevent the loss of oxidized proteins
1) Harvest cells in TNEB + Protease Inhibitor cocktail. Keep your samples ON
ICE at all times during the harvest
2) Sonicate samples
3) Add ½ of the sample into a vial containing appropriate volumes of DTT to
have a final DTT concentration of 50mM
4) Remove 50µl from the other ½ of the sample and use it for a protein assay
a. Add the remainder of the this sample to equal volumes of LB/BME
b. Heat block the sample for 10min at 95ºC
c. Use this sample for Western blotting
5) Determine protein concentrations to assure equal loading of Western blot and
Oxyblot samples. The protein assay should be performed following the
termination of the Oxyblot procedure outlined below to assure the capture
of oxidized proteins.
-----------------------------------------------------------------------------------------------------------Flow-chart of the OxyBlot Procedure
***NOTE: The following experiments will be performed at RT.
You will use the sample that contains the 50mM DTT.
You want to work with 15-20μg of protein in each condition.
1
Derivatization Reaction
5μl Protein
Negative Control
5μl 12% SDS
5μl Protein
6% SDS
5μl 12% SDS
6% SDS
Add 10μl 1X DNPH solution
Add 10μl 1X Derivatization solution
Shake well and incubate
at RT for 15min
Add 7.5μl Neutralization solution
Add 7.5μl Neutralization solution
Samples are ready for loading onto gel or
storage at 4ºC for up to 7 days.
-----------------------------------------------------------------------------------------------------------Important Notes about the Oxyblot Procedure
1) You can treat more than 5µl of protein per reaction tube. For this, adjust
the reagent volumes accordingly.
a. Example: You can use 10µl lysate and double all reagent volumes
(i.e., 10µl 12% SDS, 20µl 1x DNPH solution, 20µl 1x Derivatization
solution, 15µl Neutralization solution). This will allow you to run 2
Oxyblots from the same sample.
2) Ideally, it is best to perform the reaction right after lysing the cells.
However, samples containing 50mM DTT can be frozen at -20ºC. For best
results, do NOT store samples longer than one month.
3) Once the Oxyblot procedure has been terminated, samples can be stored
at 4ºC or alternatively, samples can be aliquated and stored at -20ºC. In
2
either case, samples should be allowed to come to RT prior to running
them on a gel.
JS 01.06.09
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