Posts Tagged ‘Protocols’

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.

Making sure you are in control of your gene expression analysis

 :: Posted by American Biotechnologist on 09-27-2011

Learn which factors affect gene expression analysis and how Bio-Rad Laboratories can help.

Novel technique reveals both gene number and protein expression simultaneously

 :: Posted by American Biotechnologist on 09-22-2011

Researchers have discovered a method for simultaneously visualizing gene number and protein expression in individual cells. The fluorescence microscopy technique could permit a detailed analysis of the relationship between gene status and expression of the corresponding protein in cells and tissues, and bring a clearer understanding of cancer and other complex diseases, according to researchers who led the study.

The new technique is called the fluorescent in situ gene protein assay. It combines traditional fluorescent in situ hybridization (FISH) with the in situ proximity ligation assay, which is capable of resolving individual protein molecules.

Read the rest of this entry »

Improving the statistic significe of your Western Blots

 :: Posted by American Biotechnologist on 09-08-2011

Western Blotting is probably one of the most ubiquitous techniques in the molecular biology lab and relatively easy to perform. Yet many of us have been frustrated with statistically insignificant results or protein bands that appear either too dark or too light to quantitate.

Well, do not despair! There are many things you can do to help improve the quality of your blots and increase your likelihood of obtaining statistically significant results!

In the video below, you will learn about the many factors affecting western blot analysis (such as detection limit and dynamic range limitations of film and overloaded gels) and what can be done to improve your chances of success.

The presentation was given by Bio-Rad Laboratories Field Application Specialist Dr. Sean Taylor as part of an intimate customer training. Some of the references in the presentation may be specific for that particular customer but the general information contained in this presentation is highly valuable to all molecular biology labs.

The bacterial industrial revolution

 :: Posted by American Biotechnologist on 08-31-2011

George Church, the talented genetic professor, has made headlines once again. We are very fond of George Church and have written about him and his work several times in the past (see: George Church: The Father of Personalized Genomics, New tools for rewriting the code of life and A Scientific Legend’s Approach to Solving Problems and Developing Technologies).

The latest article, appearing in The Boston Globe, talks about Dr. Church’s approach to synthetic biology and his “broad brush” approach of editing bacterial genomes to devise powerful new technologies.

To read more click here. H/T to Genomeweb for the find.