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Archive for the ‘Proteomics’ Category

Three important points about gel equilibration

 :: Posted by American Biotechnologist on 01-19-2012

So you’ve isolated your protein, ran them on a gel and now you’re ready to transfer them to a membrane to begin western blotting. Sounds simple, right? Not so fast. Don’t forget to equilibrate your gel prior to beginning your transfer. Gel equilibration generally involves rinsing the gel in diH2O and soaking it in transfer buffer for approximately 15 min. While it may sound simple, (and it truly is), it is a step that might make the difference between an ugly blot and one that is publication worthy.

Below are some points to consider about gel equilibration:

  1. Gel equilibration removes contaminating electrophoresis buffer salts. If not removed, these salts increase the conductivity of the transfer buffer and the amount of heat generated during transfer.
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  3. Equilibration also allows the gel to adjust to its final size prior to electrophoretic transfer. Gels shrink or swell to various degrees in the transfer buffer depending on the acrylamide percentage and the buffer composition.
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  5. Equilibration is not necessary (i) when the same buffer is used for both electrophoresis and transfer (for example, native gel transfers), or (ii) when using rapid semi-dry transfer systems such as the Trans-Blot® Turbo™ system (consult the user manual for the system you are using).

To learn more tips and tricks, download the Protein Blotting Guide from Bio-Rad Laboartories.

A new technology for studying protein interactions within the brain

 :: Posted by American Biotechnologist on 12-21-2011

Despite the difficulties associated with measuring in-situ protein-protein interactions in neural networks, Dr. Akira Chiba of the University of Miami recently announced that his team has embarked on a project to develop a protein interaction map within brain cells. Why have these studies been so difficult to perform until now and what does Dr. Chiba have that will make him successful? The answer lies in the small size of neural proteins and the technical limitations associated with even the highest resolution microscope.

Using a a custom- built 3D FLIM (fluorescent lifetime imaging microscopy), Chiba’s team has been able to spatially and temporally quantify fluorescently tagged protein-protein interactions in genetically modified fruit flies.

According to Chiba, “collaborating fluorescent chemistry, laser optics and artificial intelligence, my team is working in the ‘jungle’ of the molecules of life within the living cells. This is a new kind of ecology played out at the scale of nanometers—creating a sense of deja vu 80 years after the birth of modern ecology.”

Thanks to the University of Miami for this story.

Watch the video below, (already set to the appropriate frame…just press play), as Karl Svoboda of the Howard Hughes Medical Institute explains how FLIM works.

Proteomics: The Next Frontier in Cancer Research

 :: Posted by American Biotechnologist on 12-15-2011

Now that we’ve finished our series on Proteomics Application Tips, it’s time to reward ourselves with a captivating talk by Professor Danny Hillis on Understanding Cancer Through Proteomics. Yes…he’s preaching to the choir, but it’s still fun to see our cause promoted on the “big screen.” Enjoy!

Proteomic Application Tips Grand Finale

 :: Posted by American Biotechnologist on 12-14-2011

In previous posts we provided you with several great tips for proteomic researchers (see five great tips for researchers working with proteins and more great proteomic applications tips). Today we will present you with the final in the series of application tips which are sure to improve the quality of your proteomic experiments.

  • To ensure proper and consistent visualization with a silver stain, use ultrapure water with all organic contaminants removed for the final rinse of your staining vessel. In addition, reserve that vessel exclusively for silver staining, and separate it from other glassware in your laboratory.
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  • Proteins must be soluble if they are to be separated and identified on 2-D gels. Protein insolubility (precipitation) leads to loss of sample spots and streaking on 2-D gels.
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  • Fractionation may improve your 2-D result by reducing sample complexity, improving the range of detection, and enriching low-abundance proteins. Fractionation can be performed according to many protein properties, including subcellular location, solubility, size, charge, and pI.
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  • Improve the resolution and reproducibility of 2-D gels by performing sample cleanup to remove salts, charged detergents, phenolics, lipids, sugars, and nucleic acids. Cleanup will also reduce disulfide bonds.
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  • NaCl increases conductivity, extends the time required for focusing, causes electroendosmosis, and results in uneven water distribution in the gel. In general, the tolerated concentrations of NaCl for proper in-gel rehydration and cup loading are 10 mM and 40 mM, respectively.
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  • Nucleic acids bind proteins through electrostatic interaction, thereby interfering with isoelectric focusing. Nucleic acids can also clog the pores of the acrylamide matrix. Remove nucleic acids with nucleases and ultracentrifugation in the presence of carrier ampholytes. In addition, benzonase can be used in a sample together with urea to remove DNA or RNA contamination.
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  • Insoluble material in a sample obstructs gel pores, resulting in poor focusing and severe streaking. Remove these materials by high-speed centrifugation (for example, 20,000 x g for 30 minutes at 20°C).
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  • To monitor the initial progress of the electrophoresis on the IPG strips, add up to 0.001% of Bromophenol Blue to both the rehydration and equilibration buffers.

For more great tips visit www.expressionproteomics.com

More great proteomic application tips

 :: Posted by American Biotechnologist on 11-28-2011

In a previous post we provided you with five great tips for researchers working with proteins. Today we will present you with five more tips which are sure to improve the quality of your proteomic experiments.

  • Negatively charged polysaccharides that contain sialic acid can produce horizontal streaks similar to those generated by nucleic acid contaminants. Ultracentrifugation is often sufficient to remove carbohydrates from samples.
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  • To prevent vertical streaking, limit the amount of protein added onto an IPG strip. Compensate for such decreases in sample load by using a more sensitive staining technique, such as silver staining.
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  • Reusing electrophoresis running buffer can result in poor separation and vertical streaking due to the depletion of ions and SDS in the running buffer. Avoid this practice, especially if vertical streaking is a persistent problem.
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  • Vertical streaking on second-dimension gels is often caused by gaps between the IPG strips and the gels. Ensure the second-dimension gel has a straight and level top edge, and that the IPG strip is in direct contact with the gel along its entire length.
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  • If some of the bands on your gel are not staining or appear faint, use silver stain as usual, then agitate it slowly in deionized water for 30 minutes and repeat. Then apply the silver stain again, starting with the silver reagent step. Proteins that did not stain on the first cycle will stain to full intensity.

For more great tips visit www.expressionproteomics.com