Protein Methods Group

Hand Book for Protein purification

http://www.biochem.uiowa.edu/donelso...n_handbook.pdf

Thiru,

I got a question posted:
This message is to the protein methods forum. I want to do western blotting. Is there any detection method that is as sensitive as ECL?

thanks for sharing danfive

Purifying Proteins for Proteomics (limited preview --> http://books.google.com/books?id=ain...eJtGa-HotE-Qvw

and book review --> http://www3.interscience.wiley.com/j...98930/abstract

Storage of purified proteins (originally in http://www.pepcore.embl.de/protein_p...age/index.html )

After you have invested all the hard work to express and purify your target protein, you should not forget to think about how you want to store your purified protein. The method of choice depends completely on the stability of the protein and on when and how you want to use it later on. In general it is important to avoid storage conditions that are close to the stability limits of the protein (e.g. extreme pH or pH values close to the isoelectric point of the protein). Further, you want to avoid the addition of compounds, which interfere with your application and, therefore, have to be removed prior to use.

For short term storage (up to 24 h), most proteins can be kept at 4°C.

For storage times longer than 24 h at 4°C, it may be necessary to filter sterilize the protein preparation (through a 0.22 µm filter) or to add a bacteriostatic agent (e.g. 0.1% sodium azide) to avoid bacterial growth. Note that not all proteins are stable at 4°C for longer periods.

For long term storage (more than a week), it becomes necessary to freeze the protein preparation. It is important to freeze it rapidly using liquid nitrogen or a dry ice/ethanol mixture to avoid denaturation. It is also important to freeze the solution in small aliquots to avoid repeated freezing and thawing which may reduce the biological activity or affect the structure. Several stabilizing agents can be added, such as glycerol (5-50% (w/v)), serum albumin (10 mg/ml), reducing agents (such as 1 mM DTT), and ligands (the nature and concentration depending on the nature and concentration of the target protein).

If you want to store your protein preparation for several months, you can store it at -20°C. At this temperature it is necessary to add 50% glycerol to the solution to avoid freezing.

The sample can be prepared in two ways:
• Add an equal volume of pure glycerol to the protein preparation.
• Dialyze the protein preparation against the storage buffer containing 50% glycerol. This method has the additional advantage that it results in an approx. threefold concentration.

If you need to store your protein preparation for longer periods (months to years), you should freeze it at -70°C or even in liquid nitrogen. Although it is not really necessary to add glycerol at these temperatures, the addition of 5-50% glycerol could help to keep the protein stable.
Alternative methods:
• Storage of the protein at 4°C as an ammonium sulfate precipitate.
• Storage of the protein at 4°C or lower in a lyophilized form. For the lyophilization it is necessary that the protein is dissolved in a volatile buffer (such as trimethylamine/HCl; pH range 6.8-8.8). Note that not all proteins are stable during the freeze-drying process.

I hope to add methods for proper long term protein storage, archiving and handling in the near future, when I complete training in our new Proteomics Core Lab.