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Download the Protein Blotting Guide
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While we are all familiar with the role of methyltransferase in DNA and protein modification in the nucleus, (think epigenetics with regards to DNA), this is the first time that methylation in the cytoplasm has been shown to promote protein complex formation.
The researchers first identified an enzyme which is mainly present in the cytoplasm and which methylates the amino acid lysine (Smyd2). Then they searched for interaction partners of the enzyme Smyd2
and found the heat shock protein Hsp90. The scientists went on to show that Smyd2 and methylated Hsp90 form a complex with the muscle protein titin.
According to the authors, “Titin is the largest protein in the human body and known primarily for its role as an elastic spring in muscle cells. Precisely this elastic region of titin is protected by the association with methylated Hsp90.”
In skeletal muscle cells of the zebrafish, the team explored what happens when the protection by the methylated heat shock protein is repressed. By genetic manipulation they altered the organism in such a way that it no longer produced the enzyme Smyd2, which blocked the methylation of Hsp90. Without methylated Hsp90, the elastic titin region was unstable and muscle function strongly impaired; the regular muscle structure was partially disrupted.
Click here for a link to the Genes and Development paper.
:: 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:
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.
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.
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).
:: 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!