Posts Tagged ‘2D Gel Electrophoresis’

Five great tips for proteomic researchers

 :: Posted by American Biotechnologist on 11-03-2011

Here are some great application tips from Bio-Rad Laboratories for those researchers working with proteins:

  • Generally, the best method for keeping a protein in solution is to add any combination of nonionic detergents, zwitterionic detergents, and chaotropic agents to the sample mixture. Also use reducing agents such as DTT and DTE (less than 20 mM) to decrease disulfide bond formation between proteins.

  • When working with membrane or insoluble proteins, increase the amount of SDS in the equilibration and running buffers (up to 0.2%) to allow the proteins to effectively migrate out of the IPG strip.

  • To reduce the amount of SDS in samples generated by preparative SDS-PAGE, substitute the elution buffer with one that does not contain SDS.

  • Nucleic acid contamination is a common cause of horizontal gel streaking. Treat samples with nucleases to remove nucleic acids prior to isoelectric focusing.

  • Never heat samples in urea-containing buffers. The urea rapidly breaks down to carbamic acid and carbamylates the proteins, modifying their charge. Urea breakdown and subsequent protein carbamylation is the cause of charge trains on 2-D gels. A charge train is a series of spots on a 2-D gel that are of different pIs and the same size.

For more great tips visit www.expressionproteomics.com

An innovation in proteomics

 :: Posted by American Biotechnologist on 10-31-2011

The Protean i12 IEF cell is truly an innovation in proteomics.

To learn more about Bio-Rad’s new Protean i12 IEF Cell visit www.bio-rad.com/i12

Strategies for proteomics sample preparation

 :: Posted by American Biotechnologist on 10-25-2011

Two-dimensional (2-D) gel electrophoresis is a popular and proven separation technique for proteome analysis. The 2-D procedure is straightforward: Proteins are first separated according to their isoelectric point (pI) by isoelectric focusing (IEF) and then by their molecular weight by SDS-PAGE. For most researchers, 2-D gel electrophoresis is easy to learn, because advances in immobilized pH gradient (IPG) technology have eliminated the need for tricky and tedious IEF in ampholyte gel gradients. Nevertheless, problems with smearing, streaking, and poor resolution and reproducibility tend to leave researchers dissatisfied with the results of 2-D experiments. These common compalints are often due to improper sample preparation.

One of teh most undervalued aspects of the 2-D process, sample preparation prior to the first-dimension IEF separation contributes significantly to the overall reproducibility and accuracy of protein expression analysis. Some important considerations include:

  • Care must be taken to prevent protolysis during protein extraction, and proteins must be solubilized in a buffer that is compatible with IEF
  • Contaminants such as salts and detergents must be removed to ensure successful separation
  • Fractionation is essential to reduce protein sample complexity when analysis of subpopulations or low-abundance proteins is required

Without proper sample preparation, protein precipitations, gel streaking, and overall poor resolution are often the unfortunate result.

Click on this link to learn some great strategies for proteomic sample preparation.

Click to learn about Bio-Rad’s new Protean i12 IEF Cell.

Bio-Rad launches first IEF System with independently powered lanes for more reproducible focusing

 :: Posted by American Biotechnologist on 10-21-2011

Bio-Rad Laboratories, Inc. announces the launch of the PROTEAN i12 IEF system, the industry’s only isoelectric focusing (IEF) system that is designed to simultaneously run up to 12 immobilized pH gradient (IPG) strips in 12 independently programmed lanes.
For the best 2-D gel electrophoresis results, researchers optimize their sample preparation and first-dimension isoelectric focusing conditions to ensure adequate separation of complex samples. Because the optimal IEF conditions for different protein samples can vary significantly, each set of conditions must be tested separately. This can be a time-consuming and tedious process.

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Transfer Efficiency: Influence of the Gel Structure

 :: Posted by American Biotechnologist on 10-17-2011

With its proprietary transfer buffer, the Trans-Blot Turbo system generates very fast transfer even for high molecular weight proteins. However, as indicated previously, the gel composition, i.e. the acrylamide – bis-acrylamide network density, influences the transfer efficiency. A protein can more easily move out of the gel during the transfer if it is located in a portion of gel that has the widest pore structure. As proteins above 150 kD are always located on the first top part of a gel, the most efficient transfer of those large proteins is achieved when using gradient gel with a concentration of 4% of acrylamide – bis-acrylamide at the top of the gel. The transfer efficiency of proteins from an Any kD homogeneous acrylamide gel and a 4-20% gradient gel is illustrated.

Qualitative transfer efficiency comparison of HMW proteins on homogeneous acrylamide % and gradient gel. Precision Plus Protein™ Unstained standard and E. Coli homogenate (20 μg) were run on both Criterion TGX Any kD Stain-Free and 4-20% gels at 300V for 18 min. The total protein content was detected with the Stain-Free technology using the Gel-Doc EZ imaging system. The gels were then transferred with the Trans-Blot Turbo system with the 7 min preset program using the Trans-Blot Turbo PVDF transfer packs. The total protein content remaining in the gel is detected with the Stain-Free detection, using the same exposure parameters as used with the gels before the transfer for reliable comparison. The content of protein was also detected with the Stain-Free detection on the membrane. Even if most of the proteins are transferred in 7 min, the gradient gel contains less HMW proteins than the homogeneous gel after the transfer.