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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. Each Odyssey Loading Indicator is a single 28 kDa recombinant protein directly labeled with a near-infrared fluorescent dye. You can detect the 700 nm version of OLI in the 700 nm channel of your imaging system and the 800 nm version of OLI in the 800 nm channel.

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.

Evaluate Consistency

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

Check Uniform Transfer

Transfer variability can alter quantitative data without your knowledge. To check uniform transfer from gel to membrane, a loading control is necessary. An Odyssey Loading Indicator can reveal issues like bubbles or other transfer artifacts in the 28 kDa range.

Assess Stable Expression

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 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

(A) OLI and Housekeeping Proteins
800 nm Channel

(B) REVERT™ Total Protein Stain
700 nm Channel

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 Total Protein Stain was detected in the 700 nm channel. Targets were detected in Jurkat lysate, which had varying levels of Etoposide treatment.



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 Total Protein Stain. Both (A) tubulin and (B) COX IV signals varied in response to treatment, while REVERT Total Protein Stain signals and OLI signals remained constant. These quantitative data indicate the expression of both housekeeping proteins was affected by Etoposide treatment.

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.

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