Revert 700 Total Protein Stain for Western Blot Normalization

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Make Western blot normalization more accurate and reliable with Revert 700 Total Protein Stain, a membrane-based (post-transfer) normalization strategy that stains all protein in your sample. No special reagents, equipment, or gels are required with Revert 700 Total Protein Stain, so it is compatible with your Western blot protocol.

Use the Gold Standard for Western Blot Normalization

Total protein staining is considered the gold standard for Western blot normalization. Revert 700 Total Protein Stain provides linear signal over a broad range of sample concentrations and is compatible with subsequent Western blot immunodetection methods.

  • Quick and Compatible. Stain total protein in less than ten minutes on either PVDF or nitrocellulose membranes.
  • Accurate Normalization. With a wide linear range of 1 - 60 μg, it’s easy to detect Revert 700 stain and your target in the same linear range for accurate normalization.
  • Reliable Analysis. Unlike housekeeping proteins, biological variation won’t affect total protein normalization with Revert 700 total protein stain.

Revert 700 Total Protein Stain Workflow

Normalization Strategies

Accurate, Reliable Western Blot Normalization with Revert Stain

Revert 700 Total Protein Stain works with your existing Western blot workflow, without needing any additional reagents or special gels. Staining takes less than ten minutes. (Figure 1).

Figure 1. Rapid staining with Revert 700 Total Protein Stain. Revert 700 Total Protein Stain provides highly efficient protein staining on nitrocellulose or Immobilon®-FL PVDF membranes in under 10 minutes. 10 μg of Jurkat cell lysate was loaded in each lane and separated in 4-12% NuPAGE Bis-Tris gels, transferred to nitrocellulose or PVDF membrane, and then imaged with the Odyssey® CLx Imaging System in the 700 nm channel.

Accurate Normalization with Revert Total Protein Stain

Use this stain to normalize accurately, by correcting for lane-to-lane variation in loading and transfer of target protein in your quantitative Western blots. Revert 700 Total Protein Stain provides a linear response over a wider range of cell lysate concentration than some commonly-used housekeeping proteins (Figure 2).

Figure 2. Revert 700 Total Protein Stain has a wider linear range than housekeeping proteins. A linear response is required for quantitation. Above 20 μg of cell lysate, housekeeping proteins began to saturate and did not exhibit a linear signal response. Revert 700 remained linear from 1 to 60 μg of cell lysate. C32 cell lysate was separated in 4-12% Bis-Tris gels and transferred to nitrocellulose membrane. The relative fluorescence intensity is the average of triplicate values.

Reliable Analysis for Quantitative Western Blots

Biological variation won’t affect total protein normalization with Revert. While validation of stable expression levels is highly recommended for housekeeping proteins, Revert total protein stain is a consistent internal loading control that you don’t need to validate. (Figure 3).

Figure 3. Revert 700 Total Protein Stain is a reliable, effective alternative to housekeeping proteins for Western blot normalization. Jurkat cell lysate stimulated with TPA was used to detect an increase in pERK expression. (A) Target signals were normalized to Revert 700 Total Protein Stain, which was detected in the 700 nm channel of the Odyssey CLx Imaging System. (B) Housekeeping protein normalization with tubulin, detected in the 800 nm channel. (C) Normalized results for Revert stain and tubulin were similar for pERK detection.

Frequently Asked Questions

Should I stain my Western blot membrane with Revert 700 Total Protein Stain before or after I dry the membrane?

You can use Revert 700 on a membrane that was kept wet or dry following transfer. To use on a dry membrane, briefly wet the membrane in water prior to adding the Stain Solution. For PVDF membranes, rinse first in methanol, followed by water.

Can I reuse Revert 700 Total Protein Stain?

Revert 700 is intended for a single use and reusing the stain on additional membranes is not recommended. Reusing the stain will affect normalization accuracy.

Does Revert 700 interfere with antibody binding or the detection of my Western blot target?

Revert 700 is compatible with all subsequent Western blot immunodetection procedures and does not interfere with antibody binding.

How sensitive is Revert 700?

Revert 700 is intended for staining total protein in individual lanes and has a linear detection range of 1-60 μg when used with cell lysate. However, individual proteins are easily detectable at low nanogram amounts.

After staining my membrane with Revert 700, I am not seeing any band development.

Here are some possible causes:

  • Transfer was compromised. Check to see if your molecular weight ladders transferred.
  • Membrane was not rinsed with water prior to staining.
  • Methanol was not added to the Stain Solution.

I stained my membrane with Revert 700 Total Protein Stain and am getting very high background. Do you have any tips?

Prepare the Stain Solution by adding 30 mL of methanol prior to use.

How should I store Revert 700 Total Protein Stain and what is the shelf life?

We recommend storing Revert 700 Total Protein Stain at room temperature where it is stable for 6 months from the date of receipt.

Can I strip and reprobe Western blots stained with Revert 700?

Yes, you can strip and reprobe the membrane for subsequent detection of another target in the 800 channel.

Can I use Revert 700 on a blot that was stripped?

The signal will be weak, so we do not recommend using the Revert 700 signal to normalize the reprobed target.

Can I stain my blot with Revert 700 after blocking the membrane?

No, Revert 700 will stain blocking buffer proteins that bind to the membrane. This signal will significantly interfere with accurate detection of the protein in the sample.

Can Revert 700 Total Protein Stain be detected by other imagers?

For the best results, use Odyssey imaging systems, which are designed for superior near-infrared detection. Other imagers capable of detecting signals in the 700 nm channel can also be used with the stain. Alternatively, white light imagers can detect Revert 700 Total Protein Stain. However, the linear and dynamic range will be reduced.

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