Odyssey Loading Indicators Ensure Sample Loading Consistency

Odyssey® Loading Indicators

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Odyssey® 28KD Loading Indicator – 800 nm

Odyssey® 28 KD Loading Indicator – 700 nm

Evaluate Sample Loading Consistency and Uniformity with Odyssey Loading Indicators

Odyssey Loading Indicators (OLI) provide a simple, convenient method to evaluate the consistency of sample loading volume across gel lanes, as well as the uniformity of Western blot transfer.

It is essential to have consistent sample loading between gel lanes for comparative and quantitative Western blot analysis. By adding equal amounts of an Odyssey Loading Indicator, you can determine if loading was consistent or if a pipetting error occurred.

  • Evaluate Consistency. Ensure samples were equally loaded in each lane.
  • Check Uniform Transfer. Confirm that transfer from gel to membrane was uniform.
  • Assess Stable Expression. Help determine if a housekeeping protein is stably expressed and can be used for Western blot normalization.

Note: Accurate normalization relies on an internal loading control, an endogenous protein used as an indicator of sample concentration. Since the OLI is an exogenous protein, it functions as an external loading control, and cannot be used for normalization. However, you can use OLI to help check if your internal loading control is stably expressed.

Visit our Western blot Normalization page for more information on Western blot normalization strategies.

Assess Stable Expression of Housekeeping Proteins for Normalization

If you’re using a housekeeping protein to normalize Western blot data, you must validate that its expression is constant across all samples and is unaffected by experimental conditions. As an exogenous protein, the Odyssey Loading Indicator is not subject to up- or down-regulation as a function of experimental sample treatment. A stable signal for an OLI across several lanes indicates that any signal variation for the housekeeping protein was not caused by inconsistent sample loading.

"'House-keeping' proteins should not be used for normalization without evidence that experimental manipulations do not affect their expression."

Journal of Biological Chemistry's submission guidelines

Membrane Data Membrane Data
Figure 1. Detection of OLI 800. (A) Odyssey Loading Indicator (OLI) was detected simultaneously with the housekeeping proteins COX IV and tubulin in the 800 nm channel with the Odyssey CLx Imaging System. (B) Revert™ 700 Total Protein Stain was detected in the 700 nm channel. Targets were detected in Jurkat lysate, which had varying levels of Etoposide treatment.
Membrane Data Membrane Data
Figure 2. Assessing stable expression of tubulin and COX IV with OLI. The housekeeping protein signal intensity was plotted, along with signal intensities of OLI and Revert 700 Total Protein Stain. Both (A) tubulin and (B) COX IV signals varied in response to treatment, while Revert 700 Total Protein Stain signals and OLI signals remained constant. These quantitative data indicate the expression of both housekeeping proteins was affected by Etoposide treatment.
Membrane Data Membrane Data
Figure 3. Stable Odyssey Loading Indicator, stable COX IV. Jurkat cells were treated with increasing amounts of TPA, stimulating the target pERK (Orange, 800 nm). OLI (Green, 800 nm) was used to assess the stability of expression of the housekeeping protein COX IV (Blue, 700 nm). Standard deviations for the triplicate values of each treatment are plotted as error bars.