Removal of Interfering Substances

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Overview

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.

Interfering Substances

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

Products for Contaminant Removal

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
-
-
-
Removal of unincorporated nucleotides from DNA fragments >20 bases
-
-
-
Removal of primers and primer-dimers from PCR products >200 bp
-
-
-
-
Buffer exchange
(restriction fragments, PCR products, enzyme reactions, sequencing templates)
-
-
DNA sequencing
reaction mixture cleanup**
-
-
-
Riboprobe cleanup***
-
-
-
-
Desalting of antibody, enzyme, and protein solutions
-
-
Purification of proteins of moleular weight >6,000
-
-
-
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.

Troubleshooting

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

References

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

Related Content

Literature
Number Description Download
3096 Sample Preparation: Tools for Protein Sample Extraction, Cleanup, Fractionation, and Depletion Brochure, Rev B Click to download
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
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