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 3