Related Experiment Video
Updated: Jul 30, 2025
Analyzing Tumor and Tissue Distribution of Target Antigen Specific Therapeutic Antibody
Published on: May 16, 2020
Knockout validation of LAMP2A antibodies for immunostaining in human cancer cells
Xun Zhou1, Vera Shirokova2, Vitaliy O Kaminskyy1
1Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
Abstract:
LAMP2A is the rate-limiting factor of chaperone-mediated autophagy (CMA), a unique selective protein degradative pathway. To date LAMP2A antibodies are not knockout (KO)-validated in human cells. We have recently generated human isoform-specific LAMP2A KO cells, and here we assessed the specificity of select commercial LAMP2A antibodies on wild-type and LAMP2A KO human cancer cells. While all tested antibodies were suitable for immunoblotting, the anti-LAMP2A antibody (ab18528) is likely to exhibit an off-target reactivity in immunostaining approaches using human cancer cells, and alternative antibodies, which seem more appropriate, are available.
Insights
This study validates chaperone-mediated autophagy (CMA) related LAMP2A antibodies in human cells. One antibody showed off-target reactivity in immunostaining, suggesting alternatives for accurate research.
Area of Science:
- Cell Biology
- Molecular Biology
- Autophagy Research
Background:
- Chaperone-mediated autophagy (CMA) is a selective protein degradation pathway.
- LAMP2A is the rate-limiting factor in CMA.
- LAMP2A antibodies lack knockout (KO)-validation in human cells.
Purpose of the Study:
- To assess the specificity of commercial LAMP2A antibodies in human cells.
- To validate LAMP2A antibodies using newly generated human isoform-specific LAMP2A KO cells.
- To identify reliable antibodies for studying CMA.
Main Methods:
- Generation of human isoform-specific LAMP2A knockout (KO) cancer cell lines.
- Testing commercial anti-LAMP2A antibodies on wild-type and KO cells.
- Immunoblotting and immunostaining techniques were employed.
Main Results:
- All tested LAMP2A antibodies were suitable for immunoblotting.
- One specific anti-LAMP2A antibody (ab18528) demonstrated potential off-target reactivity in immunostaining.
- Alternative, more specific antibodies for immunostaining were identified.
Conclusions:
- LAMP2A antibody validation is crucial for accurate CMA research.
- The antibody ab18528 may lead to misinterpretation in human cancer cell immunostaining.
- Researchers should consider validated alternatives for reliable LAMP2A detection in immunostaining applications.
More Related Videos
10:29Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence
Published on: August 14, 2019
09:01Integrate Imaging Flow Cytometry and Transcriptomic Profiling to Evaluate Altered Endocytic CD1d Trafficking
Published on: October 29, 2018