Total Protein Detection

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Overview

Total protein staining provides an image of the protein migration pattern in a gel or on a blot. This information helps determine transfer efficiency and the molecular weight, relative quantity, and other properties of the transferred proteins. This section provides an overview of total protein stains, including anionic dyes (such as amido black, Coomassie blue, Ponceau S, and Fast Green), fluorescent stains, and colloidal gold stains and discusses the advantages and disadvantages of these stains. It also provides protocols for total protein staining and troubleshooting tips for various problems in staining procedures.

Related Topics: Immunodetection.

Considerations for Protein Detection

When performing total protein blot staining, note that:

  • Protein standards are useful for monitoring transfer efficiency and serve as molecular weight markers for calibration of blot patterns. Refer to protein standards section for available Bio-Rad protein standards.
  • Polyacrylamide gels shrink during staining, so comparison of an immunologically probed membrane to a stained gel is not practical. To determine the exact location of a specific antigen in relation to other proteins, compare two blotted membranes, one that has been probed with an antibody and the other stained for total protein.

Total Protein Detection

Total protein detection. Blot stained with SYPRO Ruby blot stain showing the total protein pattern of an E.coli lysate containing an over-expressed GST fusion protein on the blot.

Comparison of total protein staining methods.

Method Sensitivity   Advantages Disadvantages Imaging
Anionic dyes (Ponceau S, Coomassie Brilliant Blue R-250, amido black, Fast Green FCF) 100–1,000 ng Inexpensive, rapid Low sensitivity, shrink membrane Photography with epi-illumination or reflectance densitometry
Fluorescence 2–8 ng Sensitive, mass spectrometry-compatible Fluorescence detection system required Fluorescence visualization with UV, LED epi-illumination, or laser scanning
Stain-free (SFX) 2–28 ng Rapid – no additional staining or destaining required Special gels and imaging equipment required Gel Doc™ EZ system
Colloidal gold (enhanced) 100 pg–1 ng Very sensitive, rapid; optional enhancement increases sensitivity Expensive Photography with epi-illumination or reflectance densitometry

Anionic Dyes

The first techniques developed for total protein staining of blotted membranes used the same anionic dyes commonly used for staining proteins in polyacrylamide gels. These dyes include amido black (Towbin et al. 1979), Coomassie (Brilliant) Blue R-250 (Burnette 1981), Ponceau S, and Fast Green FCF (Reinheart and Malamud 1982). Of these:

Amido Black — destains rapidly in acetic acid/isopropanol solution and produces very little background staining. Amido black may interfere with downstream immunodetection.

Coomassie Brilliant Blue — may show high background staining, even after long destaining procedures, and is not compatible with subsequent immunodetection.

Ponceau S and Fast Green — are compatible with downstream immunodetection methods, and Fast Green can be easily removed after visualization to allow subsequent immunological probing.

These dyes are easy to prepare and they stain proteins quickly, but they are relatively insensitive when compared to other stains (see table above).

Fluorescent Stains

Fluorescent stains such as SYPRO Ruby and Deep Purple provide highly sensitive detection of proteins on blots as well as in gels. SYPRO Ruby blot stain allows detection as low as 2 ng. After staining, target proteins can be detected by colorimetric or chemiluminescence immunodetection methods, or analyzed by microsequencing or mass spectrometry with no interference from the protein stain.

Stain-Free Technology

You can visualize proteins separated by gel electrophoresis using various staining procedures or stain-free technology. Select visualization techniques based on the capabilities of the imaging equipment used in your lab and the demands of your applications.

Colloidal Gold Stain

Colloidal gold is an alternative to anionic dyes that provides detection sensitivities rivaling those of immunological detection methods (Moeremans et al. 1987, Rohringer and Holden 1985). When a solution of colloidal gold particles is incubated with proteins bound to a nitrocellulose or PVDF membrane, the gold binds to the proteins through electrostatic adsorption. The resulting gold-protein complex produces a transient, reddish-pink color due to the optical properties of colloidal gold. This gold-protein interaction is the basis for total protein staining with colloidal gold as well as for specific, immunogold detection (see Immunogold Labeling)

Troubleshooting

Comparison of total protein staining methods.

Problem Cause          Solution
Colloidal gold total protein stain — high background The blocking step was insufficient or was omitted
  • Block with 0.3% Tween 20 in TBS using 3 washes of 20 min each
Contamination occurred during electrophoresis or transfer
  • Discard and remake the gel and transfer solutions
  • Replace or thoroughly clean contaminated fiber pads if a tank blotter was used
Excessive amounts of protein were loaded on the gel or too much SDS was used in the transfer buffer. Proteins can pass through the membrane without binding and recirculate through a tank blotting system
  • Reduce the amount of protein on the gel or SDS in the transfer buffer
  • Add a second sheet of membrane to bind excess protein
The colloidal gold stain solution was contaminated
  • Use a separate, clean plastic container to store previously used reagent in the refrigerator
  • Discard any reagent that has a viscous sediment at the bottom of the bottle
  • If the solution is no longer dark burgundy but light blue, discard it. The stain is contaminated with buffer salts, which react with the gold solution, causing nonspecific precipitation of the reagent onto the membrane
The development step was too long
  • Overnight development may slightly increase sensitivity but may also increase background. Reduce development step to 1–2 hr
Colloidal gold total protein stain — low sensitivity The incubation time was insufficient
  • Increase the incubation time for detection of low-level signals. Overnight incubation is possible, although background staining can increase
Transfer was incomplete
  • See "Troubleshooting Electrophoretic Transfer" here
The stain was exhausted, as evidenced by the loss of the dark burgundy color and longer staining times
  • Discard the reagent
Buffer salt contamination has occurred; the solution is light blue instead of dark burgundy
  • Discard the reagent
Anionic dyes — high background Destaining was insufficient
  • Increase the number and duration of washes with the destaining solution
The dye solution was too concentrated
  • Prepare new solution
Anionic dyes — low sensitivity Anionic dye stains do not detect protein bands below ~100 ng
  • Use a more sensitive stain such as colloidal gold stain or a fluorescent stain
  • Increase the sample load
Fluorescent blot stains — low sensitivity Proteins with low hydrophobicity
  • Only highly hydrophobic proteins will retain enough SYPRO stain to be visible on a membrane. SDS is stripped off proteins during transfer, resulting in very little retention of the SYPRO stain on most proteins
Incorrect excitation and emission settings were used
  • Refer to the product literature for correct excitation wavelengths and emission filters

References

Burnette WN (1981). "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112, 195–203.

Moeremans M et al. (1987). The use of colloidal metal particles in protein blotting. Electrophoresis 8, 403–409.

Reinhart MP and Malamud D (1982). Protein transfer from isoelectric focusing gels: the native blot. Anal Biochem 123, 229–235.

Rohringer R and Holden DW (1985). Protein blotting: detection of proteins with colloidal gold, and of glycoproteins and lectins with biotin-conjugated and enzyme probes. Anal Biochem 144, 118–127.

Towbin H et al. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350–4354.

Related Content

Literature
Number Description Download
5781 Evaluation of the Criterion Stain Free Gel Imaging System for Use in Western Blotting Applications, Rev A Click to download
2895 Protein Blotting Guide, Interactive PDF, Rev B Click to download
2032 Western Blotting Detection Reagents Brochure, Rev F Click to download
Number Description Options
6216 Detection Buffer Formulations Click to download
6217 Total Protein Detection Click to download
6218 Blot Stripping & Reprobing Click to download
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