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.
- Determining the Linear Range for Quantitative Western Blot Detection
- Housekeeping Protein Validation Protocol
- Housekeeping Protein Normalization Protocol
- Pan/Phospho Analysis For Western Blot Normalization
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.
Do you have a question about your Western blot normalization strategy, the importance of replicates. or how publication requirements for Western blots have been changing? Contact us and let our experts help!