Oxidative Stress Defense (Catalase, SOD1, TRX, smooth muscle Actin) Western Blot Cocktail (ab179843)

Overview

  • Product name

    Oxidative Stress Defense (Catalase, SOD1, TRX, smooth muscle Actin) Western Blot Cocktail
  • Sample type

    Cell Lysate, Tissue Homogenate
  • Species reactivity

    Reacts with: Human
    Does not react with: Mouse, Rat
  • Product overview

    This Oxidative Stress Defense Western Blot Cocktail is designed to determine the relative abundance of several important proteins involved in the protection of cells against oxidative stress and the regulation of reactive oxygen species (ROS). Reactive oxygen species’ are produced naturally in cells as byproducts of the metabolism of oxygen as well as in response to various environmental stresses including UV radiation, pollutants, and heat exposure. Additionally, ROS levels can be altered by disease and injury, including cancer, neurodegenerative disease, cardiovascular disease, ischemia, stroke and aging. Reactive oxygen species also play an important role in cell signaling, a process called redox signaling. The regulation of ROS within cells is important for maintaining a proper homeostasis.


    Superoxide dismutase 1 (SOD1) scavenges harmful superoxides (O2-) within cells protecting them from harmful oxidation of lipids, proteins and nucleic acids. Its altered expression levels have been linked to Down’s syndrome, ALS and various cancers. Similarly, the hydrogen peroxide(H2O2) scavenging enzyme, catalase, also regulated ROS concentrations within cells by reducing H202 into less reactive O2 and water. Thioredoxin is a small enzyme (12kDa) that facilitates the reduction of other enzymes via cysteine thiol-disulfide exchange. Thioredoxin is used by cells to reduce ROS amounts and in redox signaling processes. Finally, alpha smooth muscle actin was included in the cocktail as a loading control. Widely expressed, smooth muscle actin is involved in cell structure and motility.


    These four readouts are easily resolved by western blot given their different molecular weights. Because they are all rabbit monoclonal antibodies, an anti-rabbit secondary should be used for detection.


     


    Expected and observed MWs:



    • Catalase: 60 kDa

    • Smooth Muscle Actin: 42 kDa

    • Superoxide Dismutase 1: 16 kDa

    • Thioredoxin: 12 kDa


     


    WB Notes:



    • - WB samples should be heated to 95°C for 5 minutes in sample buffer before loading.

    • - Suggested working concentration is 1X for primary antibody cocktail.

    • - Suggested dilution buffer is 5% milk/PBS+0.05%Tween 20.


     


    The cocktail contains 50% glycerol, can be stored at -20C. No aliquoting necessary.

  • Notes

    Related products

    Review the oxidative stress marker and assay guide to learn about more assays for oxidative stress.

  • Tested applications

    Suitable for: WBmore details

Properties

Applications

Our Abpromise guarantee covers the use of ab179843 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB Use at an assay dependent concentration.

Antibody cocktail should be diluted to 1X in appropriate dilution buffer before use.  WB samples should be heated to 95°C for 5 minutes in sample buffer before loading.

Suggested dilution buffer is 5% milk/PBS+0.05%Tween 20.

Images

  • Densiometric analysis of a western blot using ab179843 was used on various cell types to determine the relative amounts of catalase, superoxide dismutase 1 and thioredoxin.

    25 ug of each cell lysate was loaded per lane after heating for 5 minutes at 95°C.

    Lane 1: HepG2

    Lane 2: HeLa

    Lane 3: HDFn

    Lane 4: HL60

    Lane 5: Jurkat

    Lane 6: MCF7

    Lane 7: Hek293T

    Secondary: HRP-conjugated Anti-Rabbit IgG

  • WB lysate sample was heated at 95°C for 5 minutes before loading. Performed under reducing conditions.

    All blocking and antibody incubation steps were done in 5% milk in PBST.

    Developed using the ECL technique.

    Exposure time: 1 minute.

    Sample: HepG2 Cell Lysate – 25 µg/lane

    Lane 1: Anti-Catalase antibody
    Lane 2: Anti-Smooth muscle actin antibody

    Lane 3: Anti-Superoxide dismutase 1 antibody

    Lane 4: Anti-Thioredoxin antibody

    Lane 5: ab179843 Oxidative Stress Defense WB Cocktail

    Secondary: HRP-conjugated Anti-Rabbit IgG

     

References

This product has been referenced in:

  • Svarcbahs R  et al. Removal of prolyl oligopeptidase reduces alpha-synuclein toxicity in cells and in vivo. Sci Rep 8:1552 (2018). Read more (PubMed: 29367610) »
See 1 Publication for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

oxidative stress defense

Excellent Excellent 5/5 (Ease of Use)
Abreviews
isolate mitochondria from human fibroblast cell.
Actin is missing due to mito isolation
Strong band has been shown for catalase and TRX in fibroblast.
SOD1 showed other type of cells but not fibroblast cells

Abcam user community

Verified customer

Submitted Jan 24 2018

Abreviews
Blocking with 5% BSA in TBST for 1h at RT-> primary antibody (1:250 dilution) overnight at 4C -> HRP-conjugated anti-rabbit secondary antibody (1:10000) for 1h at RT-> Detection with ELC (30 second exposure)
line 1: fetal skeletal muscle (day 120 of gestation)
line 2: fetal skeletal muscle (day 165 of gestation)

Dr. Soon-Ok Kim

Verified customer

Submitted Apr 11 2017

Answer

Here are the answers to your questions:

The lysates tested with the cocktail during development were extracted with lauryl maltoside. We would expect RIPA extraction to yield similar results, but it has not been tested. Regardless of extraction buffer, this cocktail requires the heating of WB samples to 95ºC for minutes. The bands will not resolve correctly if the sample is not heated.

The antibody concentrations were optimized so that the signal was approximately equal between all bands. This varies depending on expression levels, so some bands may be stronger than others. The dilutions from the original vial of antibodies at the 1X working concentration are as follows:

Anti-Catalase: 1:3000

Anti-SOD1: 1:3000

Anti-TRX: 1:20,000

Anti-Smooth muscle Actin: 1:3000

IMR32 samples have not been tested with this cocktail. Only the cell lines shown on the datasheet were tested.

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