Protein Gel Imaging

Protein stains are used to analyze electrophoretically separated proteins while the proteins are still in the gel. The most widely used method of electrophoresis is SDS-PAGE. Before SDS-PAGE, protein samples are denatured using an ionic detergent, such as SDS, and heat. Reducing agents, such as beta-mercaptoethanol, are often used to eliminate disulfide bonds in the proteins. Then the denatured and reduced lysate is run through a polyacrylamide gel to separate the proteins by size using electrophoresis.

During electrophoresis, an electrical current is applied to migrate the negatively charged proteins through the gel. Because smaller proteins travel through the gel more easily than larger proteins, smaller proteins will run further through the gel. A wide variety of fluorescent and colorimetric stains can be used to detect the proteins after electrophoresis.

Typical Protein Gel Imaging Workflow

Add tracking dye to lysate

tracking dye + lysate

Load protein samples and marker in vertical SDS-PAGE system

loading samples

Negatively charged proteins migrate through the gel

migrating proteins

Smaller proteins migrate further than larger proteins

migrating proteins

SDS-PAGE gel after Coomassie blue staining

coomassie blue staining

Acquire image using an Odyssey Imager

coomassie blue staining

Fluorescent Staining

Fluorescent stains can be imaged in both near-infrared (NIR) and visible fluorescent wavelengths. Fluorescent staining is generally considered to be more sensitive than colorimetric staining. There are many fluorescent stains that can be imaged on Odyssey® Imagers, including:

Figure 1: Coomassie Blue
Coomassie Blue
Figure 1: Spyro Ruby
Spyro™ Ruby
Figure 1: Pro-Q Diamond Phosphoprotein Stain
Pro-Q™ Diamond Phosphoprotein Stain
Figure 1: Spyro Orange
Spyro™ Orange

Colorimetric Staining

Colorimetric stains, such as Coomassie blue or silver stains, can be imaged using the Odyssey M Imager. Silver stain offers more sensitivity than Coomassie, but the protocol can be complex. Coomassie stains, in contrast, are a simpler, quicker, and more affordable way to detect proteins. There are many colorimetric stains that can be detected on an Odyssey M Imager, including:

Coomassie Blue image
Coomassie Blue
Silver Stain image
Silver Stain

Coomassie Staining

One of the most popular methods to stain a protein gel is with Coomassie blue. Coomassie blue protein stains are anionic and bind non-specifically to proteins. Coomassie blue protein stains can be used for sensitive, quantitative protein detection in gels, with a linear range from ~10 ng to 20 µg.1

Coomassie blue protein stain is a strong 700 nm fluorophore. Fluorescence of Coomassie stain is induced upon protein binding. Coomassie-stained protein gels can be imaged on any of the Odyssey Imagers using NIR fluorescence with comparable sensitivity to SYPRO™ Ruby, but at a lower cost.1

As shown in Figure 1, the Odyssey M Imager allows you to image Coomassie blue protein gels in many channels, including fluorescent and colorimetric channels.

figure 1 RGB Trans figure 1 700 nm Fluorescent
figure 1 630 Trans
Figure 1. An example protein gel stained with Coomassie SimplyBlue™ SafeStain imaged in multiple channels. The gel was imaged using the RGB Trans, 630 nm Trans, and 700 nm Fluorescent channels of an Odyssey® M Imager, respectively. Lanes 1 – 3 contain an unstained molecular weight marker. Lanes 4 – 7 were loaded with 10 µg C32 whole cell lysate. Lanes 8 – 11 were loaded with hemagglutinin protein spiked into 10 µg C32 lysate. Lanes 12 – 15 contained only the hemagglutinin protein.

Silver Staining

Silver staining is another popular method to identify proteins within gels after electrophoretic separation. Silver staining is a highly sensitive method of colorimetric detection that works by incubating a gel in silver nitrate to bind silver particles to proteins. The silver particles stain the proteins various shades from yellow to brown to black, which can then be imaged using the RGB Trans or 470 nm Trans channels of the Odyssey M Imager, as shown in Figure 2.

While silver staining offers high sensitivity, the protocol used can be complex and may generate high background.

figure 2
figure 2
Figure 2. An example protein gel stained with silver stain imaged in multiple channels. The gel was imaged using the RGB Trans and 470 nm Trans channels of an Odyssey M Imager, respectively. Lanes 1 – 3 contain an unstained molecular weight marker. Lanes 4 – 7 were loaded with 0.5 µg C32 whole cell lysate. Lanes 8 – 11 contained only the hemagglutinin protein. Lanes 12 – 15 were loaded with hemagglutinin protein spiked into 0.5 µg C32 lysate.

Uses for Protein Gel Staining

Protein gels are often used to assess protein purity and to detect products in forced degradation studies for the development of therapeutics. A protein gel can also be used to detect glycoproteins and phosphoproteins or can be run separately to supplement the findings of an immunoblot.

Glycoprotein Detection

Visualization of all protein bands in the gel is possible with Coomassie staining, or glycoproteins may be imaged using a glycoprotein-specific stain, such as Pro-Q™ Emerald. Glycoprotein bands with a known molecular weight can be identified by referencing a molecular weight marker lane on the gel or membrane. Shift changes due to digestions can be documented when comparing treated to non-treated samples, as shown in Figure 3.

figure 3
Figure 3. Example of glycoprotein detection with Coomassie stain on gels. Fetuin was digested with various glycoprotein specific enzymes, PNGaseF (P), neuraminidase (N), and O-glycanase (O). The reactions were separated on a 7.5% NEXT Gel and stained with Coomassie. The total protein stain of the gel confirms the PNGase F activity due to the protein shift in molecular weight.
figure 4
Figure 4. An example protein gel stained with Pro-Q™ Emerald Glycoprotein Stain. Gel was imaged using the 520 nm fluorescent channel of an Odyssey M Imager. Lanes 1 – 3 were loaded with 10 – 2.5 µg C32 whole cell lysate. Lanes 5 – 8 were loaded with a serial dilution of CandyCane™ Glycoprotein Molecular Weight Standards.

Phosphoprotein Detection

Phosphorylated protein bands may be detected within a gel without requiring transfer to a blotting membrane using Pro-Q™ Diamond Phosphoprotein Gel Stain or other phosphoprotein stains.

figure 5
Figure 5. An example protein gel stained with Pro-Q™ Diamond Phosphoprotein Stain. Gel was imaged using the 520 nm fluorescent channel of an Odyssey M Imager. Lanes 1 and 15 contain Chameleon® Duo Pre-Stained Protein Ladder. Lanes 2 – 4 were loaded with 10 – 2.5 µg MCF7 whole cell lysate. Lanes 5 – 7 were loaded with 10 – 2.5 µg MCF7 Etoposide-treated lysate. Lanes 8 – 14 were loaded with a serial dilution of PeppermintStick™ Phosphoprotein Molecular Weight Standards.

See some published examples of protein gel imaging

View publications

References

  1. Luo, S., Wehr, N.B., and Levine, R.L. (2006). Quantitation of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast Green. Anal. Biochem., 350(2), pp. 233-238. DOI: 10.1016/j.ab.2005.10.048

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