Validate Housekeeping Proteins Before Using Them for Western Blot Normalization


Why is it important to validate housekeeping proteins before using them for Western blot normalization?

Housekeeping proteins (HKPs) are routinely used for Western blot normalization. For common HKPs (such as actin, tubulin, or GAPDH), stable protein expression is generally assumed. However, expression of several HKPs is now known to vary in response to certain experimental conditions, including cell confluence, disease state, drug treatment, and cell or tissue type. Because HKP normalization uses a single indicator of sample loading, changes in HKP expression will introduce error and may alter data analysis and interpretation.

In the instructions to authors for the Journal of Biological Chemistry, they state:

Before using a housekeeping protein for Western blot normalization, it is critical that you validate that its expression is constant across all samples and unaffected by your experimental context and conditions, especially if you have plans to publish.

How do I validate housekeeping proteins?

The Housekeeping Protein Validation Protocol gives you step-by-step instructions on how to validate a housekeeping protein for use as an internal loading control. It also provides information on how to analyze the data in Image Studio™ software (download free Image Studio Lite) and how to quantitate housekeeping proteins. Detailed calculations and information on how to interpret the data will allow you to be confident in your validation process – and make the right decision for your Western blot normalization strategy.

When you have completed your housekeeping protein validation and have determined that the HKP expression is unaffected by your experimental conditions, you can use the Housekeeping Protein Normalization Protocol and proceed with using your validated HKPs for Western blot normalization and quantitative analysis.

There you go! Unlike what other vendors may be telling you, you CAN use housekeeping proteins for Western blot normalization – as long as you validate that their expression is not changing under your experimental conditions. Download your copies of Housekeeping Protein Validation Protocol and Housekeeping Protein Normalization Protocol and get started today.

Other Protocols to Support Western Blot Normalization

LI-COR has several other protocols to help you meet new stringent publication guidelines and requirements. These are detailed protocols and include information on how to analyze and interpret your data.

With all of these protocols and our scientific experts, we can help you collect accurate, reliable Western blotting data. You will be confident in your results and your conclusions. When you submit your data for publication, you will be confident that you are meeting even the toughest publication standards. Protocols are also available in an online format at protocols.io

The Gold Standard for Western Blot Normalization: Total Protein Staining



In the instructions to authors for the Journal of Biological Chemistry, they state:

While you have choices for your Western blot normalization strategy – you can still use housekeeping proteins as long you have validated that their expression is not changing – total protein staining detection is becoming the “gold standard” for normalization of protein loading.

After transfer, but prior to immunodetection, the membrane is treated with a total protein stain to assess actual sample loading across the blot. Because this internal loading control uses the combined signal from many different sample proteins in each lane, error and variability are minimized. This antibody-independent method corrects for variation in both sample protein loading and transfer efficiency, and monitors protein transfer across the blot at all molecular weights. The figure at the left shows that REVERT Total Protein Stain provides highly efficient protein staining on nitrocellulose or Immobilon®-FL PVDF membranes in under 10 minutes. Complete figure legend.

REVERT™ Total Protein Stain is a near-infrared fluorescent membrane stain used for total protein detection and normalization. REVERT staining is imaged at 700 nm, and fluorescent signals are proportional to sample loading.

The REVERT Total Protein Stain Normalization protocol describes how to use REVERT Total Protein Stain for Western blot normalization and quantitative analysis. It includes step-by-step instructions on how to use REVERT stain. There is also detailed information on normalization calculations, analysis of replicates, and data interpretation.

Replication is an important part of quantitative Western blot analysis and is used to confirm the validity of observed changes in protein levels. Biological and technical replications should both be done, since they are both important but meet different needs.

LI-COR has several other protocols to help you meet publication guidelines and requirements. In all of them, key factors for success, data analysis and interpretation are covered as well as links to additional educational resources.

With these protocols and our scientific experts, we can help you collect accurate, reliable data that will meet even the toughest publication standards. Protocols are also available in an online format at protocols.io

Download your copy of REVERT Total Protein Stain Normalization protocol and use the gold standard to determine your protein loading concentrations. Let us help you be confident in the Western blotting data you submit for publication.

The Importance of Detecting in the Combined Linear Range for Western Blots



In the instructions to authors for the Journal of Biological Chemistry, they state:

What is the linear range of detection?

In quantitative Western blot analysis, the linear range of detection is the range of sample loading that produces a linear relationship between the amount of target on the membrane and the band intensity recorded by the detector.

Within the linear range of detection, band intensity should be proportional to the amount of target. A change in target abundance should produce an equivalent change in signal response. At the upper and lower ends of the linear range, this proportional relationship is lost. Band intensity no longer reflects the abundance of target, and quantification is not possible.

Quantitative Western blot analysis is only accurate if the target protein and internal loading control can both be detected within the same linear range – a range that must be determined experimentally for each target and loading control. The combined linear range is then used to determine how much sample should be loaded to produce a linear signal response for both the target protein and the internal loading control.

Are YOU detecting your target protein and your internal loading control in the combined linear range?

How is the combined linear range determined?

Help has arrived! The protocol “Determining the Linear Range for Quantitative Western Blot Detection” from LI-COR explains how to use serial dilutions of sample protein to determine the linear ranges of detection for a target and internal loading control, and choose an appropriate amount of sample to load for quantitative Western blot analysis.

This protocol also explains key factors for success, required reagents, data analysis and interpretation. Two methods for determining the linear range are included in the protocol:

  • Determining the Linear Range for a Target Protein and REVERT™ Total ProteinStain. Follow these instructions if total protein staining of the membrane will be used as the internal loading control for quantitative Western blot normalization.
  • Determining the Linear Range for a Target Protein and a Housekeeping Protein. Follow these instructions if a housekeeping protein will be used as the internal loading control for quantitative Western blot normalization. This method also applies to normalization with a pan-specific antibody for analysis of phosphorylation or other post-translational modifications.

LI-COR has several other protocols to help you get published. In all of them, key factors for success, data analysis and interpretation are covered as well as links to additional educational resources.

With these protocols and our scientific experts, we can help you collect rock-solid data that will meet even the toughest publication standards. Protocols are also available in an online format at protocols.io

Download your copy of Determining the Linear Range for Quantitative Western Blot Detection so that you can accurately determine the linear range for your quantitative western blot detection. Let us help you be confident in the Western blotting data you submit for publication.