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Download the Protein Blotting Guide
Download the Stem Cell Guide for Life Science Researchers
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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.
:: 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.
Now, we are proud to present you with a 43 page protein blotting guide put together by Bio-Rad Laboratories. The guide is organized into two parts which cover the theory and methods behind protein blotting. You will learn topics such as methods and instrumentation, the difference between various membranes and tranfer buffers, the ins and outs of transfer conditions, detection and imaging and a host of different blotting and detection protocols.
The guide is fairly technical and is appropriate for both novice and advanced users alike.
:: Posted by American Biotechnologist on 11-10-2011
Angiogenesis is a fundamental process required for multiple physiological and pathological events. It is also a hallmark of over 50 different disease states, including cancer, rheumatoid arthritis, cardiovascular diseases, diabetes and psoriasis. Methods developed to study these diseases are important tools for testing potential therapeutics. These methods include both in vivo and in vitro assays. In vivo assays are considered the most informative because of the complex nature of vascular responses to test reagents. However these assays are often costly and laborious. In contrast, in vitro assays can be carried out expeditiously, are less expensive, and are easier to interpret. Often, these in vitro assays provide maximum benefits when developed as multivariate index assays where the data of multiple assays yield a composite profile of clinically relevant protein biomarkers.
Bio-Rad has developed a multiplex Bio-Plex Pro human cancer biomarker panel that measures angiogenesis biomarker in diverse matrices including serum, plasma, cell culture supernatant and many other sample types. Using this assay, it is possible to quantify multiple angiogenesis targets n a single well of a 96-well microplate in just three hours, using as little as 12.5ul of serum or plasma.