Overview

  • Product name

    PDH Rodent Immunocapture Kit
  • Sample type

    Cell culture extracts, Tissue Extracts
  • Assay type

    Quantitative
  • Species reactivity

    Reacts with: Mouse, Rat
  • Product overview

    The pyruvate dehydrogenase complex performs the decarboxylation of pyruvate into acetyl CoA, a critical function in metabolism, linking glycolysis and oxidative phosphorylation in mitochondria. The enzyme is composed of multiple copies of three enzymes: pyruvate dehydrogenase (E1), dihydrolipoamide transacetylase (E2) and dihydrolipoamide dehydrogenase (E3). The E1 enzyme is a tetramer of two alpha (PDHA1) and two beta (PDHB) subunits.

    Abcam ab133988 PDH Rodent Immunocapture Kit is suitable to immunocapture PDH complex from rodent tissue homogenates and whole cells lysates. Traditional immunoprecipitation methods usually result in co-elution of the antibody heavy and light chains that may co-migrate with relevant bands, masking important results. The Abcam PDH immunocapture Kit resolves this issue by immobilize the PDH capture antibody onto protein G-agarose beads. The kit includes optimized buffers and reagents for sample preparation and PDH binding and recovery, which shorten the protocol and minimize handling and mixing.

  • Tested applications

    Suitable for: IPmore details

Properties

  • Storage instructions

    Store at +4°C. Please refer to protocols.
  • Components 200 µg
    10 X Extraction Buffer 1 x 1ml
    10 X PBS 1 x 10ml
    10 X Wash Buffer 1 x 10ml
    Glycine Elution buffer 1 x 1ml
    Immunocapture Antibody Coupled Agarose Beads 1 x 100µl
    Neutralization buffer 1 x 1ml
    SDS Elution Buffer 1 x 1ml
  • Research areas

  • Alternative names

    • Pyruvate dehydrogenase

Applications

Our Abpromise guarantee covers the use of ab133988 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
IP Use at an assay dependent concentration.

Images

  • For all lanes: 10 µL antibody coated beads was mixed with 1 mg rodent sample and incubated for 3 hours. Captured PDH complex was eluted with SDS elution buffer and half volume of the eluted PDH complex was loaded on the gel. Gel was stained with Colloidal Coomassie Blue.

    Lane 1: Ladder
    Lane 2: PDH complex of RHH (rat heart homogenate)
    Lane 3: PDH complex of MHH (mouse heart homogenate)
    Lane 4: PDH complex of 1mg H4IIE (rat) whole cell lysate
    Lane 5: PDH complex of 1mg 3T3 (mouse) whole cell lysate

Protocols

References

This product has been referenced in:

  • Basak NP  et al. Alteration of mitochondrial proteome due to activation of Notch1 signaling pathway. J Biol Chem 289:7320-34 (2014). Read more (PubMed: 24474689) »
See 1 Publication for this product

Customer reviews and Q&As

Question
Answer

Thank you for your inquiry.

I heard back from the lab with the following recommendations:

The beadsare pre-coated with an anti-PDH antibody, and offered as a slurry in PBS/azide (100ul beads in 1ml PBS/azide).
1. You will first work on sample preparation, i.e. prepare mouse skeletal muscle homogenate, determining the protein concentration of the homogenate from a small aliquot of the homogenate, and then bring the concentration to 5.5mg/ml with 1X PBS.
2. The second step is to solubilize the homogenate with 10X extraction buffer following the protocol. (i.e. add 1 volume of 10X extraction buffer to 9 volume of homogenate @ 5.5mg/ml). after 20 minutes incubation, the lysates will be collected using centrifugation.
3. Add 0.5 to 1mg protein lysates to 5-10 ul beads(50-100 ul beads slurry, suspend the beads thoroughly before pipeting. (optional) Add 500ul to 1ml PBS to make sure the beads are suspended well.
4. After 3 hour incubation, beads will be collected and washed. Transfer the beads to a new tube before you elute the target protein to help to reduce the background, but this step is not crucial and is not included in the protocol.
5. Elute protein with proper volume of either SDS elution buffer or Glycine elution buffer andfollow the protocol.
6. Neutralize and wash the beads for future reuse.



I hope this information helps. Please contact us with any other questions.

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