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Download the Protein Blotting Guide
Download the Stem Cell Guide for Life Science Researchers
:: Posted by American Biotechnologist on 01-12-2012
So you think that protein transfer for western blotting is a piece of cake? Consider these important tips before proceeding:
Use only high-quality, analytical grade methanol. Impure methanol can increase transfer buffer conductivity and yield a poor transfer.
In many cases, ethanol can be substituted for methanol in the transfer buffer with minimal impact on transfer efficiency. Check this using your samples.
Do not reuse transfer buffer since the buffer will likely lose its ability to maintain a stable pH during transfer.
Do not dilute transfer buffers below their recommended levels since this decreases their buffering capacity.
Do not adjust the pH of transfer buffers unless specifically indicated. Adjusting the pH of transfer buffers can result in increased buffer conductivity, manifested by higher initial current output and decreased resistance.
Increasing SDS in the transfer buffer increases protein transfer from the gel but decreases binding of the protein to nitrocellulose membrane. PVDF membrane can be substituted for nitrocellulose when SDS is used in the transfer buffer.
Addition of SDS increases the relative current, power, and heating during transfer, and may also affect antigenicity of some proteins.
Increasing methanol in the transfer buffer decreases protein transfer from the gel and increases binding of the protein to nitrocellulose membrane.
:: 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!
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.