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Frequently Asked Questions

What does it mean if the Odyssey® Loading Indicator band intensity is different in some of my lanes?

This suggests a pipetting error, meaning that different amounts of sample volume were loaded in these lanes.

Can I use the Odyssey Loading Indicator to normalize my Western blot data?

No, only internal loading controls can be used for Western blot normalization. Internal loading controls are endogenous proteins that are unaffected by experimental conditions and used as an indicator of sample concentration. The Odyssey Loading Indicator is an exogenous protein, so it does not vary to the same degree with sample concentration. Get the Normalization Handbook to learn how to use internal loading controls.

What are the applications for the Odyssey Loading Indicator?

The Odyssey Loading Indicators provide a simple and convenient method for determining consistency of sample loading between gel lanes and Western blot transfer. You can also use a Loading Indicator to validate stable expression of housekeeping proteins for Western blot normalization and to correct for errors in sample loading.

Can Odyssey Loading Indicators be detected by other imagers?

Yes, but for the best results, use Odyssey imaging systems, which are designed for superior near-infrared fluorescence detection. Other imagers capable of detecting signals in the 700 nm or 800 nm channel can also detect the Loading Indicators.

Do the Odyssey Loading Indicators interfere with antibody binding or the detection of my Western blot target?

The Loading Indicators are compatible with all subsequent Western blot immunodetection procedures and do not interfere with antibody binding.

I’m using the Odyssey Loading Indicator to validate stable expression of my housekeeping protein. The Loading Indicator signal is consistent in every lane, but there are noticeable differences in the housekeeping protein signal in my control versus treated samples.

  1. The expression of the housekeeping protein is affected by experimental conditions. Consider using total protein staining for normalization with REVERT™ Total Protein Stain.
  2. The same volume was loaded in each lane but there are differences in the total amount of protein in each lane of the blot. Use a protein concentration assay to make sure that all samples are loaded with an equivalent amount of total protein.

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