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:: 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.
:: 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.
:: Posted by American Biotechnologist on 08-10-2011
In the past, we’ve discussed the importance of selecting appropriate reference genes for your qPCR experiment (also see point 7 of the MIQE guideline checklist). This means that it is important to select genes that do NOT exhibit any changes in expression under the treatment conditions you are studying. This is easier said than done!
“Once upon a time” everyone used either beta actin, 18s, or gapdh as reference genes. Their expression never changes, right? Wrong! So which genes should you choose? If you try to figure it out using previous papers, how do you know that they’ve chosen the correct genes? If you run a few genes side-by-side and try to compare their expression both under treatment and control, which one should you set as the baseline and which one can you say is for sure moving (it’s all relative isn’t it)?
One of my twitter friends told me that she uses six reference genes in her qPCR experiments. I used to use two. That got me thinking…how many reference genes does the “average” lab use? Please help satisfy my curiosity by participating in the poll below!
The presentation goes through the intricacies of primer design, ideal amplicon characteristics, troubleshooting with a thermal gradient, how reagents can impact your experiment, primer titration, assay sensitivity, standard curves, the affect of PCR inhibitors, how scale affects reproducibility, throughput and speed.