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Download the Protein Blotting Guide
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:: Posted by American Biotechnologist on 11-15-2011
We found the amateur home video on protein blotting and western blot analysis posted on teachinhawaii’s YouTube channel. The video is a decent step-by-step demonstration of how to perform protein blotting and western blot analysis. It is appropriate for novice or first-time users. The video shows how to do protein blotting with Bio-Rad’s Mini-Trans Blot or Criterion Protein Blotting Systems. Of course, faster protein blotting can now be perfomed in under 3 minutes with Bio-Rad’s Trans-Blot Turbo Transfer System (as opposed to the 1-2 hours suggested in this video).
We are in the process of collecting protein blotting home videos. If you are aware of an interesting video, please let us know.
:: 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.
:: Posted by American Biotechnologist on 11-01-2011
Proteins are literally the movers and the shakers of the intracellular world. If DNA is the film director, then they are the actors. And much can be learned about cell function – and dysfunction – by watching proteins on the move.
:: 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.