Loading control antibodies are used in Western blotting and other protein assays to ensure that the amount of protein loaded onto a gel or membrane is consistent across samples. These antibodies bind to proteins that are typically expressed at constant levels in all cells or under specific experimental conditions, providing a reference point for normalizing protein expression data.

Purpose and Importance

  • Normalization: Loading controls are essential for accurate quantification of target proteins. Variability in sample loading or transfer can lead to misleading results if not corrected for.
  • Validation of Experimental Conditions: Ensuring that the protein expression levels are comparable across samples helps validate experimental conditions and technical consistency.

Methodology

  • Western Blotting:
    • Sample Preparation: Equal amounts of protein from each sample are loaded onto an SDS-PAGE gel.
    • Blotting and Detection: Proteins are transferred to a membrane (e.g., PVDF or nitrocellulose) and probed with antibodies against both the target protein and the loading control.
    • Normalization: Band intensities of the target protein are normalized to the loading control to account for variations in sample loading and transfer efficiency.
  • Other Protein Assays:
    • Immunofluorescence: Loading controls may be used to ensure consistent cell staining and protein localization across samples.
    • ELISA: Loading controls help validate sample preparation and ensure reliable quantification of target proteins.

Technical Considerations

  • Selection of Loading Control:
    • Consistency Across Samples: The chosen loading control should be stably expressed and not affected by experimental treatments or conditions.
    • Relevance to Study: For specific studies, especially involving different tissues or cell types, select a loading control that is relevant to the experimental system.
  • Validation:
    • Check for Expression Levels: Verify that the loading control protein's expression is not altered under the experimental conditions being tested.
    • Cross-Validation: Using multiple loading controls can provide additional assurance of accurate normalization.
  • Technical Issues:
    • Cross-Reactivity: Ensure that the loading control antibody does not cross-react with other proteins or experimental conditions.
    • Consistent Transfer and Detection: Ensure that the blotting and detection processes are consistent to avoid variations in band intensities.

203.48 203.48000000000002 USD
AffiAB® Anti-Beta-Gal Antibody
CAT# AFG-BMG-0005
Size: 100 µl
248.20 248.20000000000002 USD
1,573.55 1573.55 USD
1,573.55 1573.55 USD
1,199.15 1199.15 USD
2,667.50 2667.5 USD