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Immunoblotting PKC-delta: a cautionary note from the bench
Vitalyi O Rybin1, Susan F Steinberg
1Department of Pharmacology, College of Physicians and Surgeons, Columbia University, 630 W. 168th St., New York, NY 10032, USA.
American Journal of Physiology. Cell Physiology
|October 21, 2005
Summary
Commercial antibodies for protein kinase C-delta (PKC-delta) can misrepresent total protein levels due to phosphorylation masking. Careful antibody selection is crucial for accurate signaling pathway research.
Area of Science:
- Cellular signaling
- Immunoblotting techniques
- Phosphoprotein analysis
Background:
- Antibodies are vital for studying signaling pathways, but their recognition properties are often uncharacterized.
- Commercial antibodies' performance can vary, impacting research accuracy.
- Understanding antibody behavior is critical for reliable results in cell signaling studies.
Purpose of the Study:
- To evaluate the characterization of commercially available antibodies used in signaling pathway research.
- To investigate the recognition properties of an anti-PKC-delta antibody in cardiomyocytes.
- To identify antibody features affecting immunoblot analysis of phosphoproteins.
Main Methods:
- Immunoblot analysis of PKC-delta in resting and PMA-treated cardiomyocytes.
- Treatment with a PKC inhibitor (GF-109203X) and acid phosphatase.
- Evaluation of anti-PKC-delta-pY(311) antibodies from different manufacturers.
Main Results:
- A BD Transduction Laboratories anti-PKC-delta MAb recognized PKC-delta only in resting cells, masked by phosphorylation in PMA-treated cells.
- This antibody artifactually indicated decreased total PKC-delta protein upon phosphorylation.
- Specific anti-PKC-delta-pY(311) antibodies detected stimulus-induced phosphorylation, with variations in performance between manufacturers.
Conclusions:
- The epitope recognized by the BD Transduction Laboratories anti-PKC-delta MAb is masked by phosphorylation.
- This antibody is unsuitable for comparing PKC-delta levels in resting versus activated states.
- Findings highlight the need for thorough antibody validation in phosphoprotein immunoblotting, potentially indicating a broader issue in phosphoprotein analysis.