Mini-Protean TGX Stain-Free Precast Gels from Bio-Rad

February 26, 2013

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Quality of Results

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University of Washington
Pharmacology
Graduate Student

Representative samples imaged in stain-free system. 3 second exposure time. Lane 1: Bio-Rad Precision Plus Protein Dual Color Ladder, Lane 2: Bio-Rad Precision Plus Unstained Ladder, Lanes 3 - 6: 4 different samples

Company:

Bio-Rad

Product Name:

Mini-Protean TGX Stain-Free Precast Gels, Any kDa, 10 wells, 30 ul

Catalog Number:

456-8123

Image

SDS-PAGE is a mainstay for many labs, especially those interested in protein function. In our lab, we work with high molecular weight proteins on a regular basis, as well as low molecular weight proteins, so we use gradient gels. We use these gels for Western blotting, coomassie staining, and silver staining. In the particular experiments in which I am using the Bio-Rad Stain Free gels, I am purifying protein complexes whose subunits range from 200 kDa to 10 kDa and using SDS-PAGE to estimate yield and purity of the complex. The purified complexes range in concentration from 0.1 mg/mL to 3 mg/mL. This product appealed to me because the gel running time was only 30 min, and protein bands were instantly visible by UV after gel sandwich disassembly.

Experimental Design and Results Summary

Application

Western blotting, coomassie staining, silver staining

Starting Material

I purified several different GST fusion proteins that were over expressed in E. Coli. These proteins were purified on glutathione resin and eluted in Laemmli sample buffer containing SDS.

Protocol Overview

I mixed the purified samples (in buffer containing 20 mM HEPES, 200 mM NaCl, 1 mM EDTA, and 2 mM DTT) with 2x Laemmli Sample Buffer containing 5% beta-mercaptoethanol to a total volume of 30 ul. I loaded these samples into the gel (10 well, AnykDa), which was in a Bio-Rad Mini-Protean running box, with Tris-Gly running buffer. I ran the gel for 30 min at 200 V, after which I disassembled the gel sandwich and placed the gel on a UV imaging system that included a camera. After “activating” the gel by exposing it to UV, I was able to image the gel with short exposures (<5s). After this imaging, I coomassie stained the gel.

Tips

Rinsing the gel in water prior to imaging seems to increase background, so it’s best to image prior to rinsing.

Results Summary

I was able to see well-resolved protein bands, even in low concentration samples. However, some small proteins were not visible by the stain-free method, and only appeared after coomassie staining. I later learned that this is due to a limitation of the stain-free method. The imaging of protein bands in this system relies on UV-induced chemical modification of Trp residues, and several of the proteins I was purifying had no Trp residues. These proteins were easily visualized by coomassie staining in later steps, however. Overall, this system reduced the amount of time from sample loading to imaging from approximately 2.5 hours to 30 min.

Additional Notes

The image quality using the instant UV imaging is not as high as can be obtained by more traditional staining methods, so the UV imaging serves best as a preliminary imaging method to be followed by coomassie or silver staining.

Related Categories

Image Gallery

Representative samples imaged in stain-free system. 3 second exposure time. Lane 1: Bio-Rad Precision Plus Protein Dual Color Ladder, Lane 2: Bio-Rad Precision Plus Unstained Ladder, Lanes 3 - 6: 4 different samples

Summary

The Good

Cost-effective compared to other precast gels; fast; easy to use.

The Bad

Cannot visualize proteins that do not have Trp residues; requires a UV imaging system.

The Bottom Line

If you are using precast gels, it’s definitely worth it to give these a shot. However, like all precast gels, they are expensive.

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