I am purifying a His-tagged protein from an EColi expression system. After the purification I am doing an activity assay so it is vital that I get active protein. First, it seems that as my protein concentration increases the activity of the enzyme decreases. This reminds me of a negative dominant type situation, but does this occur with soluble purified proteins? After purification I use the vortexed purified protein for my activity assay. I want to do a western blot to confirm presence of protein, so I took the remainder of my eluted protein and spun it down. Much to my surprise, there was a white pellet in my eluted protein samples. I'm not sure what this pellet is. My best guess is it is aggregated protein. It seems counter-intuitive to think that my protein is eluted in the elution buffer past the resin bed but then aggregates in the elution buffer. Any help is appreciated. Thanks :)
Among possible reasons you might want to consider:
1) If the isoelectic point of your protein is close to the Elution Buffer pH your protein will be prone to aggregation and precipitation (Try to change pH up or down at least 1.0 - 1.5 pH unit)
2) You Elution Buffer might have too high or too low salt concentration (try to make desalting or add salt on the small aliquot of your sample)
3) It's possible that this pellet is not your aggregated protein but it represents an insoluble salt derivative resulted from interaction of different salts in your Elution Buffer (try run a gel on the pellet and supernatant separately)
4) If your eluted protein has a lot of contaminating proteins it can interact with them forming aggregates (try to use additional Washing Step before elution with Imidazole concentration lower then that you use for Elution)
5) Try to avoid vortexing of your protein samples.
Hi Julie
i used to express and purify a his tagged enzyme from E Coli So yield and activity in particular were paramount. I cannot comment specifically without your actual SOP. If you can provide I will comment in a more specific and meaningful way. However I will say this:
1. If your elution conditions are wrong then an increase in protein concentration could lead to aggregation of protein and loss of activity
2. Vortexing can DEFINITELY denature proteins I WOULD NOT recommend
3. Depending on how you elute it might be possible to denature your enzyme for assay and storage purposes by using centricon columns. The important thing is to keep your protein in 10% to 50% Glycerol at a certain pH and ionic strength. I can send you an exact SOP if you are interested
Among possible reasons you might want to consider:
1) If the isoelectic point of your protein is close to the Elution Buffer pH your protein will be prone to aggregation and precipitation (Try to change pH up or down at least 1.0 - 1.5 pH unit)
2) You Elution Buffer might have too high or too low salt concentration (try to make desalting or add salt on the small aliquot of your sample)
3) It's possible that this pellet is not your aggregated protein but it represents an insoluble salt derivative resulted from interaction of different salts in your Elution Buffer (try run a gel on the pellet and supernatant separately)
4) If your eluted protein has a lot of contaminating proteins it can interact with them forming aggregates (try to use additional Washing Step before elution with Imidazole concentration lower then that you use for Elution)
5) Try to avoid vortexing of your protein samples.
Hi Julie,
Viktor is right.
I would add two things:
1. Aggregation can be slow and appear over time, which explains why your protein looks OK after elution and aggregates after a while. Among things that can promote aggregation are: concentration (so concentrate by steps, mix your protein at each step and if your protein becomes opalescent, stop right away, centrifuge the protein and keep the supernatant), temperature (complex effect but the rule when you don't know the protein is keep at 4°C for short times, -20°C or -80° for longer times), freeze-thaw cycles, so either keep your protein all the time at 4°C and make new preps frequently or flash-freeze small aliquots of your protein with a cryoprotectant like sucrose or glycerol. At the beginning, use a fresh aliquot for every assay.
2. Sometimes imidazole by itself causes aggregation over time. When I suspect this, I elute with the lowest possible imidazole concentration (200-250 mM generally) and I dilute 4-5 times in elution buffer without imidazole, and I concentrate the protein back to its original concentration.
Olivier
One more thing:
Vortexing proteins can be done but gently, in small volumes (like 100 µl in an eppendorf 1.5 ml tube), and the rule is : if it makes bubbles, it's bad!. However, if it produces a nice vortex in the liquid without any bubbles, in my hands it's OK. If you don't succeed, or for larger volumes, mix by pipetting up and down. But try first to mix sample buffer in water that way, you will see that it requires quite a lot of pipetting until you get a homogeneous solution!
Olivier
hi Julie, often the protein elution buffer is not the best one to store your protein in since they still have salt in it and might interfere with its stability. hence what we do a buffer exchange of the protein to a more complatible buffer like say a phosphate buffer. u can try a different buffer system to store your protein and see if it still aggregates or in some case precipitates out of solution.
I'd like to add that there are some general rules for handling proteins, but every protein has its individual preferences in terms of buffer, handling and storage conditions, etc. You can easily identify best conditions for your protein by nanoDSF and especially you will know which conditions or handling steps to avoid. Read more about this in the application note attached.
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