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.
Evans DR et al. (2009). Concentration of proteins and removal of solutes. Methods Enzymol 463, 97–120.
