Requirements for Internal Loading Controls

Normalization Webinar InvitationFor more information on Western blot normalization, watch these webinars:


Western blots are packed with potential sources of variability. Variability that isn’t accounted for limits reproducibility and threatens your chances for publication-quality data. Normalization corrects for variability introduced during the process of Western blotting.

So what should you do to get more reproducible data? Use an internal loading control for each blot. Internal loading controls are endogenous sample proteins that are stably expressed and unaffected by experimental conditions.

Requirements for an Effective Internal Loading Control:

  • Linear, proportional response. Signal intensity of the internal control should accurately reflect sample con¬centration and abundance of loading control over a wide range.
  • Low biological variability. Your experimental treatments should not affect the expression of your internal loading control. For example, expression of some housekeeping proteins may vary in response to experimental conditions.
  • Corrects for variation at all stages of immunoblotting. Your internal control should correct for variation that occurs throughout the Western blot process, including gel loading and transfer.
  • Compatible with immunodetection. The strategy you choose shouldn’t interfere with effective down¬stream detection of your target proteins.

For more information about internal loading controls, check out the full review article:
Western Blot Normalization: Challenges and Considerations for Quantitative Analysis

What Factors Affect Normalization?

Normalization Webinar InvitationFor more information on Western blot normalization, watch these webinars:


Do you know what factors affect normalization? Routine steps in the Western blotting process such as sample preparation, sample loading, and the detection of multiple proteins can introduce unwanted variability. You should plan to reduce error in every step of the Western blotting process. Without planning, you might get pseudo-quantitative results that don’t reflect the biology of your samples.

Sample Preparation

Blog - Sample prepThe way you prepare your samples can significantly change the results of your experiment. Even small changes in plating, cell lysis, reagent volume, and other technical details can have a surprising impact.

For example, how you lyse your cells affects protein extraction, solubilization, and modification status. The insoluble fraction may retain relevant proteins, affecting your quantitative analysis. Some experimental treatments shift fractions between soluble and insoluble.

For these reasons, it’s important to be consistent when preparing your samples. It’s also good practice to estimate the total protein concentration of each sample after preparation. Bradford, BCA, and Lowry assays are widely used to estimate the total protein concentration. Then it’s possible to adjust gel loading to the estimated protein concentration.

Sample Loading

Blog - sample loadingOverloaded gels create problems. Although strong bands may appear similar, the bands could be saturating either the membrane capacity or the dynamic range of detection. To avoid saturation and inaccurate results, run a standard curve with two-fold serial dilutions of cell lysate. You can then identify the linear range for each target protein.

Detection of Multiple Proteins

You may need to detect multiple proteins to compare relative protein levels, especially if you’re using a housekeeping protein or signaling protein to normalize. Stripping and reprobing is often used to compare different proteins on the same blot, but it can introduce error. Leftover antibodies from incomplete stripping result in artifacts. Overly harsh stripping may result in a loss of sample proteins from the membrane.

If, however, you use near-infrared fluorescent detection, there’s no need to strip and reprobe. Multiplexing is when you detect two different proteins with spectrally-distinct secondary antibodies. Multiplexing is convenient and saves time. It is also more accurate than stripping and reprobing, because no artifacts are introduced, and there’s no possibility for protein loss. With multiplexing, you can use co-migrating proteins, as well as easily identify antibody cross-reactivity.

For more details about factors that affect normalization, check out the full review article:
Western Blot Normalization: Challenges and Considerations for Quantitative Analysis