Through a series of posts I have recently shared tips and insights about improving your protein purification preparation. It included bioinformatics analysis for protein purification, affinity chromatography, Ion-exchange chromatography and Size exclusion chromatography. Now that you have successfully established a pure sample, how should you store it? And what can you do to learn about the process and retain that crucial know-how about your protein?
Analysis of your protein
Before commencing with downstream experiments, you should first evaluate the purification level of your protein and its current concentration (and decide if you want to concentrate or dilute it).
The best and cheapest way to evaluate your protein purity is through the use of SDS-PAGE analysis combined with sensitive staining (coomassie staining or better, silver staining). You can also detect minute amounts of contaminants and degradation products can established using Matrix assisted laser desorption/ionization – time of flight analysis (MALDI-TOF). While this method requires only micrograms of protein sample, it bears two downsides: a) it requires buffer exchange into a salt-free buffered solution which can affect the stability of your protein sample and b) that it might not always detect all species within the sample (depends on the ionization capability of each peptide/protein specie).
The easiest and fastest test to assess your protein concentration is through UV absorbance measurement at 280nm. An exception for such method are cases in which your proteins doesn’t absorb at this wavelength, usually due to low number of Tryptophan and/or Tyrosine residues. In such case the use of the Bradford assay (as well as other colorimtetric assays) can be the only way to work around this limitation. It is crucial, however, to either determine which protein is suitable for the calibration curve or to keep using the same protein control so you will consistently calculate your protein sample’s concentration.
Monitor your protein batch collection with Labguru
An important aspect of protein purification and characterization is to maintain your knowledge, know-how and small intricate info about your protein’s behavior. While the process of protein purification has a goal on its own, you can learn a lot about your protein’s behavior to different buffers, pH range and adaptability to changing environments, all of which should be logged and kept safe. Labguru web-based tool for managing your research has a module which is explicitly deals with keeping track of all your samples, and in our case, your protein sample. The module contains all the aspects of your protein purification process such as name, gene ID and specie source, source of expression, purification methods and much more. Description of the exact box in which your samples are located makes this module highly comfortable when you’re ready to test your purified protein. No more wasting precious time and getting a cold burns searching through a deep freezer. The Labguru’s connectivity connects all aspects of your protein purification, whether it was the reserve of the FPLC, purification methodology and any modification you’ve performed. A text free description box is the place where you can describe key aspects of the protein’s behavior (“protein sediments at low salt”) or the specific aim in which this protein should be used for (see screenshot below). An excel sheet can be downloaded from the module to expedite the upload of the information into all the fields in a quick blaze. Try it now for free!
How to maintain long term storage of proteins?
Proteins are active moieties which rotate, tumble and vibrate at room temperature constantly and at nanoseconds rate. This rapid motion within the solution can have an adverse effect on protein’s long range structural and functional stability. Thus, once you are satisfied with the protein’s purity, protein concentration and buffer composition, you should place the protein at freezing temperature as soon as possible. The low temperature (the lower the better) will keep your protein at close to stationary state and will extend the proteins longevity within the freezer. The low temperature also inhibits any microbial growth, especially if you didn’t filtered your protein sample prior to freezing it.
The lowest temperature which can be achieved in a standard biochemical lab is close to -200 degree Celsius (liquid nitrogen) though for protein storage a -80 degree Celsius is sufficiently low enough and cost effective. But, wait! Don’t go and stash your protein samples into the deep freezer just like that!
The inherent limitation of freezing an aqueous solution is the transition of water from liquid to solid phase. At this transition temperature (a few degrees above zero Celsius) water molecules interaction shifts from disorder to ordered interaction just before they are transformed into ordered hexagonal crystalline structure, swiftly affecting the hydrophilic-hydrophobic environment in which proteins are held. From the hydrophilic hugging environment, less and less water molecules hydrate the polar residues leading eventually to a decrease of the hydrophobic effect which keeps folded proteins from unfolding an exposing their hydrophobic core.
This is the reason why it is recommended to flash freeze your proteins using liquid nitrogen; in such manner the passage through the denaturation-potential temperature is so short that most protein molecules are not expected to be affected before the temperature is freezing temperature
Since frequent freeze-thawing cycles pass each time through the freezing temperature, it can lead to the denaturation of more and more protein molecules in the sample. To cope with such a problem, you should divide your protein sample into aliquots taking into account the expected down-stream experiments and the amount of protein required. In such a case, a number of thawed vials should be used or thrown to the trash after several days, according to the stability of the specific protein.
How do you treat your proteins? Have you also monitored all your batch purifications?? Leave a comment and share!