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.
Total protein staining is a direct measure of the total amount of sample protein in each lane. For each lane, the sum of all the signal intensities of all the proteins in the lane is used for normalization. This more direct approach may increase the accuracy of normalization. Unlike housekeeping proteins, total protein staining does not require validation for each experimental context. A total protein stain should produce a linear increase in signal intensity in response to increasing protein concentration. It should also correct for variation at all points in the Western blot process, including gel loading and transfer to membrane. And, it must be compatible with downstream immunodetection of your blot.
You should make sure that the signal intensity of the total protein stain is moderate, without saturation or low signal-to-noise ratios. Revert™ 700 Total Protein Stain provides linear, proportional signal across a broad range of sample concentrations.
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 on the right shows that Revert 700 Total Protein Stain provides highly efficient protein staining on nitrocellulose or Immobilon®-FL PVDF membranes in under 10 minutes.
Revert 700 Total Protein Stain is a near-infrared fluorescent membrane stain used for total protein detection and normalization. Revert 700 staining is imaged at 700 nm, and fluorescent signals are proportional to sample loading.
The Revert 700 Total Protein Stain Normalization protocol describes how to use Revert 700 Total Protein Stain for Western blot normalization and quantitative analysis. It includes step-by-step instructions on how to use Revert 700 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. Refer to Quantitative Western Blot Analysis with Replicate Samples to guide you in your experimental set up.
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 700 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.
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