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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.

7 Great Resources for HRM Analysis

 :: Posted by American Biotechnologist on 08-04-2011

Bio-Rad Laboratories recently launched the Precision Melt Supermix, which is a high-perfomance supermix for both genotyping and epigenetic analyses.

In honor of this launch, we invite you to review some of the resources (including technical notes, review articles and video tutorials) that we have posted on high resolution melt analysis. Feel free to to click on any of the links below for further details:

Working Range vs. Dynamic Range in Quantitative Immunoassays

 :: Posted by American Biotechnologist on 06-01-2011

For any quantitative immunoassay, the distinction between the dynamic range of an assay versus working range of an assay is an important consideration. Many believe the working range of an assay and the dynamic range of an assay are one and the same. However, this is not the case. Here we discuss the distinction between these two terms.

For quantitative immunoassays the dynamic range of an assay is described as the lowest to the highest concentration of an analyte that can be reliably detected by the assay. This is sometimes referred to as the lower and upper limits of detection (LLOD and ULOD, respectively). Although signal is detected, the accuracy and precision of this number may vary beyond what is acceptable to report as an accurate measure of the concentration of the target. Although still a useful measure, dynamic range is not as rigorous a measure of the true range of the assay.

For most labs, the working assay range is a more meaningful measure of the upper and lower limits of quantitation (ULOQ/LLOQ) of an assay. The working range of an assay is commonly defined as the range over which analyte concentrations can be quantitated with acceptable precision and reliability. Because this is a stricter measure and requires both sensitivity and accuracy, the working range is typically narrower than the dynamic range. However, it is a more reliable measure of the true range of concentrations that can be accurately quantitated.

As compared to the dynamic range, the values associated with working range of an assay are both precise (defined as how reproducible multiple measurements or calculations are) and accurate (defined as how close a measured or calculated quantity is to its true value).

Bio-Rad Laboratories is a leading provider of instrumention and assays for performing quantitative immunoassays. Bio-Rad’s Bio-Plex instrument is a powerful system for quantitative analysis of up to 100 different proteins, peptides, DNA fragments and RNA fragments from a single drop of sample.

To learn more be sure to view the following Bio-Plex tutorial videos posted right here on the American Biotechnologist.

A powerful system for multiplex analysis

Programming Bio-Plex Manager software

Analyzing Bio-Plex experimental data

A Revolutionary Tool for 5 Minute Protein Visualization and Analysis

 :: Posted by American Biotechnologist on 11-11-2010

A short while ago, Bio-Rad Laboratories (who we all know as experts in electrophoresis and gel documentation) came out with a revolutionary new imaging system that allows for visualization of RNA, DNA and proteins and provides publication-quality images and analysis in seconds — with just the push of a button. (For more reading on the Gel Doc™ EZ system see our previous post on the subject.) Since it’s launch, the Gel Doc™ EZ System has been well received by the research community and is proving to be a darling tool of molecular biologists everywhere.

In the attached technical bulletin, you will learn more about a new technology that dramatically reduces the time required to detect and quantify proteins in a gel, and improves the user’s ability to reproducibly validate an affinity purification procedure.

The paper describes how the Gel Doc EZ Imaging system can be used in combination with stain-free gels to reduce background noise and give superior results for non-quantitative and quantitative gel analysis. This means that bands that could not have otherwise been seen using a standard Coomassie stain can now be visualized with the Criterion Stain Free system.

If you’re interested in protein visualization in polyacrylamide gels within 5 minutes of completing electrophoreic sample separation (no staining, destaining or fiddling with imaging system settings necessary), you should definitely read this bulletin.

Rapid Validation of Purified Proteins using Criterion Stain Free Gels.

A Practical Guide to High Resolution Melt Analysis Genotyping

 :: Posted by American Biotechnologist on 10-28-2010

High resolution melt (HRM) analysis is a relatively new technique used in detecting small variations in DNA sequences between varying populations. Important applications of HRM include SNP analysis, genotyping and methylation analysis. The technique relies on quantitative analysis of the melt curve of a DNA fragment following amplification by PCR and in combination with qPCR permits the identification of genetic variation in nucleic acid sequences by the controlled melting of a double-stranded PCR amplicon. As opposed to standard melt curves which are run for routing qPCR experiments, HRM melt curves involve the collection of melt data in 0.2 degree C increments. Furthermore, in order to identify small nucleotide changes it is essential to eliminate background fluorescence from any HRM analysis. Recent advances in real time PCR equipment, software and reagents (including fluorescent dyes) has turned HRM into a robust analytical technique capable of detecting a small proportion of variant DNA in a background of wild-type sequence at sensitivities approaching 5%. Perhaps one of the most astounding accomplishments of HRM analysis is the ability to detect class IV SNPs (A>T or T>A mutations) which are extremely rare (they occur at a frequency of approximately 7% withing the Human Genome) and difficult to identify due to their small melt curve temperature shift.

The attached technical note from Bio-Rad Laboratories will provide you with a fantastic overview of HRM analysis and a detailed list of things to consider prior to embarking on HRM analytical experimentation. More specifically, the technical note will cover:

- important features required for HRM compatible instrumentation
- key features for HRM compatible software
-experimental design considerations for successful HRM analysis including: HRM-compatible saturating dyes, primer design and amplicon length and PCR reaction optimization

At the tech note mentions, HRM is a low-cost, readily accessible technique that can be used to rapidly analyze multiple genetic variants. Careful sample preparation and planning of experimental and assay design are crucial for robust and reproducible results. Following the attached guidelines will assist in the development of such assays.

HRM technical note

Sean Taylor is a Field Applications Scientist at Bio-Rad Laboratories and the primary author of this tech note. Click here for other technical resources from Sean including his video entitled “A Practical Approach to MIQE for the Bench Scientist,” and the article A Simple Solution to Chromatography for High-Purity Protein Preparations: The Modular Approach