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The combination of the hydrogen peroxide treated and untreated set is designed for use as a western blot sample when studying protein modification by oxidation and oxidative damage.
Lysates were generated from HeLa cells treated with either 10 mM hydrogen peroxide for 10 minutes or a vehicle control.
Concentration: HeLa H202 lysate, 200 µg at 2.0 mg/mL
HeLa Vehicle lysate, 200 µg at 2.0 mg/mL
Exogenous hydrogen peroxide produces oxidatively damaged cellular components and produces oxidative stress. Oxidative modification of proteins modifies the side chains of methionine, histidine, and tyrosine and forms cysteine disulfide bonds. These modifications can cause changes to a protein’s function and structure.
Dithiothreitol (DTT) has been left out of the lysates to preserve any disulfide bonds that are often affected by the presence or absence of oxidation. DTT can be added to the samples if desired.
|HeLa Hydrogen Peroxide Lysate||1 x 200µg|
|Hela Vehicle Lysate||1 x 200µg|
The hydrogen peroxide (Lane 2) and vehicle (Lane 1) treated HeLa lysates were used as protein sources for the Oxidized Protein Western Blot Kit (ab17820) that detects carbonyl groups present in the lysates. The relative abundances of the carbonyl groups were determined in the two samples.
As expected, densiometric analysis shows that the hydrogen peroxide treated lysate contains significantly more carbonyl groups created from the oxidation of amino acid residues.
An anti-2-Cys Peroxiredoxin mouse monoclonal antibody (ab16765) was used to measure the relative amounts of 2-Cys Peroxiredoxin monomer and dimer in the hydrogen peroxide (Lane 2) and vehicle (Lane 1) treated Hela lysates. Peroxiredoxin reduces oxidative species by forming a disulfide containing dimer under oxidative stress. However, if oxidation levels are too high, the reducing cysteine sites are over-oxidized, blocking the formation of the dimer. This effect can be seen in Figure 2. The Peroxiredoxin dimer amount is shown to be relatively high due to the naturally high amount of oxidative stress in Hela cells. However, when the cells are treated with hydrogen peroxide, Peroxiredoxin is over-oxidized, preventing the formation of dimers.
Over-oxidized Peroxiredoxin (PRX-SO3) was also measured directly using the anti-PRX-SO3 specific ab16830 in hydrogen peroxide (Lane 2) and vehicle (Lane 1) treated Hela lysates. The image shows that the addition of hydrogen peroxide over-oxidizes the reactive cysteine site of Peroxiredoxin, inhibiting its activity.
ab181414 has not yet been referenced specifically in any publications.
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