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Download the Protein Blotting Guide
Download the Stem Cell Guide for Life Science Researchers
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 11-03-2011
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.