Success or failure of any protein analysis depends on sample purity. Interfering substances that can negatively impact gel electrophoresis include salts, detergents, denaturants, or organic solvents (Evans et al. 2009). Bio-Rad products that can be used for contaminant removal are discussed in this section.
Related Sections: Cell Disruption, Protein Solubilization, and Sample Quantitation.
Page Contents
In addition to chemical contaminants, protein samples may contain an excess of biomolecules such as DNA or carbohydrates. Highly viscous samples indicate high DNA and/or carbohydrate content, which may also interfere with PAGE separations. In addition, solutions at extreme pH values, such as fractions from ion exchange chromatography, diminish the separation power of most electrophoresis techniques. Use one of the following methods as needed to remove these contaminants:
- Protein precipitation — the most versatile method to selectively separate proteins from other contaminants consists of protein precipitation by trichloroacetic acid (TCA)/acetone followed by resolubilization in an electrophoresis sample buffer. A variety of commercial kits can simplify and standardize laboratory procedures for protein isolation from biological samples
- Buffer exchange — size exclusion chromatography is another effective and rapid method for removing salts, detergents, and other contaminants
Bio-Rad offers kits designed for removal of salts, detergents, and other contaminants. They incorporate procedures such as precipitation and size exclusion chromatography to improve resolution of SDS-PAGE and 2-D gels. For contaminant removal Bio-Rad offers the following:
- ReadyPrep™ 2-D cleanup kit — uses a modification of the traditional TCA protein precipitation protocol. The kit offers quantitative protein recovery as well as ensures easy and reproducible removal of interfering substances
- Bio-Spin® and Micro Bio-Spin™ 6 columns (see table) — provide rapid salt removal in an easy-to-use spin-column format. Accommodating up to 100 µl of sample, these columns remove compounds <6 kD; proteins can be eluted in urea-based sample buffer for 2-D applications
Bio-Rad products that can be used for contaminant removal. Top, Micro Bio-Spin and Bio-Spin columns; bottom, ReadyPrep 2-D cleanup kit.
Column Selection Guide
| Bio-Spin 6 | Micro Bio-Spin 6 | Bio-Spin 30 | Micro Bio-Spin 30 | PCR Kleen | |
| Packed support | Special grade Bio-Gel® P-6 gel |
Special grade Bio-Gel P-6 gel |
Special grade Bio-Gel P-30 gel |
Special grade Bio-Gel P-30 gel |
Special grade size exclusion gel |
| Equilibration buffer | 10 mM Tris, pH 7.4 or SSC buffer* | 10 mM Tris, pH 7.4 or SSC buffer* | 10 mM Tris, pH 7.4 or SSC buffer* | 10 mM Tris, pH 7.4 or SSC buffer* | 10 mM Tris, 1 mM EDTA, pH 7.0 |
| Desalting of oligonucleotides >20 bases |
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| Removal of unincorporated nucleotides from DNA fragments >20 bases |
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| Removal of primers and primer-dimers from PCR products >200 bp |
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| Buffer exchange (restriction fragments, PCR products, enzyme reactions, sequencing templates) |
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| DNA sequencing reaction mixture cleanup** |
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| Riboprobe cleanup*** |
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| Desalting of antibody, enzyme, and protein solutions |
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| Purification of proteins of moleular weight >6,000 |
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| Purification of proteins of molecular weight >40,000 |
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| Bed Volume | 1.1 ml | 0.7 ml | 1.1 ml | 0.7 ml | 0.6 ml |
| Retention and recovery | 90% recovery of 20 bases or bp, 99% retention of salts | 90% recovery of 20 bases or bp, 99% retention of salts | 95% recovery of 22 bases or bp, 98% retention of ddNTPs | 95% recovery of 22 bases or bp, 98% retention of ddNTPs | 85% recovery of ≥700bp, 95% retention of primers and primer-dimers |
| Molecular weight exclusion limit, globular proteins | 6,000 | 6,000 | 40,000 | 40,000 | 8,000,000 |
| Sample volume | 50–100 µl | 10–75 µl | 50–100 µl | 10–75 µl | 25–100 µl |
| Centrifuge type | Swinging bucket | Microcentrifuge | Swinging bucket | Microcentrifuge | Microcentrifuge |
| Autoclavability | Yes | Yes | Yes | Yes | Yes |
* 150 mM NaCl, 17.5 mM sodium citrate, pH 7.0
** In Tris buffer.
*** In RNase-free Tris buffer.
| Problem | Cause | Solution |
| Laemmli sample buffer turns yellow | Sample buffer is too acidic | Add Tris base until buffer turns blue again |
| Sample very viscous | High DNA or carbohydrate content |
Fragment DNA with ultrasonic waves during cell lysis and protein solubilization Add endonucleases like Benzonase Precipitate protein with TCA/acetone (ReadyPrep 2-D cleanup kit) to diminish carbohydrate content |
Documents
TEST
| 6194 | Protein Sample Generation General Tips | Click to download |
| 6195 | Protein Sample Preparation (Human Tissue) | Click to download |
| 6196 | Protein Sample Preparation (Mammalian Tissue) | Click to download |
| 6197 | Protein Sample Preparation (Plant Leaves) | Click to download |
| 6198 | Protein Sample Preparation (Microbial Cultures) | Click to download |
| 6199 | Buffer Formulations | Click to download |