Stability is an inherent property of the protein(s) of interest and the source. Not all proteins behave similarly under the same storage conditions. For example, many nucleic acid modifying enzymes, such as restriction enzymes, can be stored for months at -20 or -80 degrees in various concentrations of glycerol but with different buffer components. However, I once worked with an enzyme from mammalian liver that was only stable while attached to an affinity column. After removing the enzyme from the column it would rapidly lose activity. The same enzyme from a different organ did not exhibit loss of activity. Both enzymes were purified to the same level as far as my analytical tools would allow me to measure. I would thoroughly examine the literature for stability characteristics of your protein or proteins of interest and pay special attention to the source of the protein(s). If you cannot find a perfect match between the literature protein(s) and the one(s) you are studying, then test all of the storage conditions to determine which formulation performs best. You can make further modifications at that point if necessary. I should also mention that storage temperature is a critical parameter to examine. The crystal structure of water is different depending upon temperature and materials in the water, e.g. buffer components. A protein suspended in a one storage buffer may be very stable at - 80 degrees C but rapidly lose activity at -20 degrees C.

Hi João,

In my experience, samples should not be freezed more than 2 years at -80oC. ELISA vs CBA, I never have tested both at the same time and compared the results, but they shoud be equivalent.

Stability is an inherent property of the protein(s) of interest and the source. Not all proteins behave similarly under the same storage conditions. For example, many nucleic acid modifying enzymes, such as restriction enzymes, can be stored for months at -20 or -80 degrees in various concentrations of glycerol but with different buffer components. However, I once worked with an enzyme from mammalian liver that was only stable while attached to an affinity column. After removing the enzyme from the column it would rapidly lose activity. The same enzyme from a different organ did not exhibit loss of activity. Both enzymes were purified to the same level as far as my analytical tools would allow me to measure. I would thoroughly examine the literature for stability characteristics of your protein or proteins of interest and pay special attention to the source of the protein(s). If you cannot find a perfect match between the literature protein(s) and the one(s) you are studying, then test all of the storage conditions to determine which formulation performs best. You can make further modifications at that point if necessary. I should also mention that storage temperature is a critical parameter to examine. The crystal structure of water is different depending upon temperature and materials in the water, e.g. buffer components. A protein suspended in a one storage buffer may be very stable at - 80 degrees C but rapidly lose activity at -20 degrees C.

I agree with the above opinions. Additionally, some proteins contain thiol group which can be affected by atmospheric oxygen. Freezing and thawing can affect this process. Analysis of said proteins by said method may or may not be affected by these bonds which can affect primary, secondary, tertiary, structure as well as functionality. Modification of post-translational modifications such as these through sample storage, frozen or not, may greatly affect what you observe. I agree, when possible, fresh is best.

It really depends on the protein...enzymes are less stable than structural proteins, but you have to judge from case to case. I have a protease that I store at -80C for years and it's still fine after 5 years. Other enzymes I store only a few months.

Hi João,

I believe you have already gotten the answer to your question: it will all depend on what you are looking for! And also within the cytokines you will observe different degrees of stability! As for ELISA x CBA, I have seen different results on equivalent samples, suggesting that each assay may maintain differently the ability to detect the proteins after storage.

I work mainly with photosynthetic RC from Rb. sphaeroides and Cyt c and my experience that frozen at -80°C they are stable for years.

Hi, in my opinión, it depends on protein structure and frozen temperature. In my experience cell lysates with proteases inhibitors could be used during 6-8 months for WB analysis without problems. Generally, I prefer to store these lysates at -40C rather tha-80.

Under the right storage conditions, proteins may be stable for many years when stored at low temperature. Many enzymes will tolerate direct freezing and thawing in buffer solutions. Others require a cryoprotectant/stabilizer like 50% glycerol. Some proteins will tolerate storage in buffer almost indefinitely at 4 deg C if concentrated (>10 mg/mL) and protected from microbial degradation. We have many samples of active protein in our freezer or fridge that have been there for years. If your proteins tolerate lyophilization, they can be stored at -20 to -80 deg C indefinitely.

The principal challenges to protein survival are concentration/aggregation of protein, and temperature-dependent pH changes that occur during the freezing or thawing stages. Some proteins tolerate this better than others. Oxygen sensitive proteins must be stored in the presence of reducing agents and the absence of metal ions to suppress thiol oxidation over time. One particularly horrible protein of ours could only be stabilized in 100 mM DTT and 50% glycerol, 1 mM EDTA at pH 6.8 or lower and could not be frozen, but would tolerate low temperature storage (without freezing) at -20 deg C.

If your protein of interest is cytokine than storage at liquid nitrogen or -80 will be sufficient for cytokine stability and reliability. Since repeated freeze thaw cycle affect any protein, small aliquot of samples under same conditions are recommended for reproducibility of the results.

Cytometric bead array (CBA) is of course a flow and bead based multiplex platform for quantifying cytokines cannot be compared with ELISA. as each of these techniques has its own strength and weakness, depending on your need it can be applied. For Instance, if your objective is to find out cytokine profile in a given situation then CBA or Bioplex are best for you or if you want to find a profile as well as to validate particular cytokine then CBA followed by ELISA will be good. Unlike DNA Microarray expression profile which will validated with RT-PCR, the validation by ELISA is not mandatory some times, again it depend on what you need to validate. TGF-beta is an example you can't use the value obtained from CBA unless and until you validate with ELISA due to nature of antibody and cross-reactivity in multiplex platform. So its all depend on your need.

Dear Colleague,

WE have been performing routine and research with many Antgens, prtoein itself. We store in -20oC for 6 months, in -80oC for up to 2 years but my personal experince is best for 1,5 years. Liquid nitrogen is another option, but you have to have very good controlled facility and expertise to work with it.

Dear Colleagues

I froze supernatant of the cell cultures at -80 C up to 2 years.

I froze tumor cells (from hematologic source) at -196 C up to a maximun of 0.5-1 year. The cells were thawed and cultured within this time-period.

If the cells were thawed later than 1 year:

1) The viability of the cells were reduced considerably.

2) In the cell cultures the rate of the dying cells were very high.

3) The cultivated cells were deformed (examination under microscope).

Best

Eva

Hi Luciana, Alex and everyone, thank you very much for all the positive answers. I think I've got what I need so far.

Go to ibdl.ca and search through the application notes on the front page will lead you to "New product evaluations" then take a look at "Evaluation of recombinant FGL2 Stability under Different Temperature Conditions" For a detailed info.

For how long can RT-qPCR samples be frozen in minus 80 degrees deep freezer without being degraded?

It seams that The data every time （duration more than 1 month）I did from the same samples changed......

I have a follow-up question, Can cytokines collected in supernatants of cell cultures be stored at -80 degrees without use of protease inhibitors?

Anything should be "dead" at -80°C, so I don't think the inhibitors would be required for the storage per se, but they may be important for the handling around (before you freeze them and between you thaw them and start working with them)

Thanks for your response Tomas.