I am attempting to purify a protein with his-tag. Current lysis method includes his-lysis buffer (pH8) with 1% triton and 2minute sonication (pulse 10seconds, rest 40 seconds)
after incubation with Co-resin, the protein seems to be present in the flow through fractions and not in the elution fractions, suggesting that the his-tag has not bound to the resin.
Experiment works with Bugbuster without sonication.
Was simply wondering if there were suggestions as to how to lysis can be done with sonication ensuring that protein binds to resin. Thank you!
Your sonication procedure seems mild, but it dosent work- You may vary time and frequency to rule out any possibility. Your experiment works with bugbuster, you can try bead beater or just freeze thaw instead of sonication.
Sonication at that rate shouldn't have any effects on the His-tag. However, you can try sonication by forcing your cell lysate in a 26G or 27G (preferably) syringe needle 10 times at least, a classic (but golden and cheap) method but does work and produce end relatively similar results.
Dear Sarah
generally sonication, if well performed, itself do not alter protein conformation and ability to bind the resin. However the choose of the right sonication cicle (combination time, pulse and break) is essential because a weak sonication can only partially extract the protein while a too storng sonication can result in increase in sample temperature that can induce protein unfolding.
In your case the cicle seems quite weak and if you perform the step in ice i'm quite sure that you do not have problem of unfolding.
Two questions:
1) Why do you use tritonX 1%? Is it your protein a membrane protein of partially soluble?
Becuase if your proten tend to aggregate is it possibile that different buffer composition may change it aggregation state, and not aggregate has his exposed and bind to the resin while if aggregate the his tag is buried into the aggreegate and it not bind to the resin.
2)Is it Bugbuster contain tritonX 1%?
because:
- Are you sure that the CO-resin IMAC tollerate 1% of Triton?
It seems to me borderline (for example Talon manual suggest amount
What is your protein? is from bacteria or mammalian?
sometimes his tag hide behind your target protein.
Manuele Martinelli We used triton 1% because We found it helped lyse the cells without the need for Bugbuster.
in all the different scenarios to lyse cells (triton, bugbuster) samples with sonication didnt have any protein in the elution fraction. It is present in soluble, flow through and the final SDS wash fraction. Seems like with sonication, the protein either doesn’t stick or isnt coming off with the elution buffer.
When tried with JUST bugbuster (no sonication), protein was present in the elution fraction.
Thank you for your help and response !
Hi Sarah,
Before setting up the experiment,, I would suggest you to optimize your experimental condition:
A. Buffer: You can chose few buufers RIPA(very strong), 0.1% NP-40 (mild), Buffer-C or any of your choice (depending upon protein localisation-cyto./nuc.)
B. Sonication condition: Fix to one condition 20X --> 1min on ice. Repeat one more time (Pulse 20 times)
C. Check WB using anti-tag (His) and anti-protein.
Observe and plan your experiment selecting the best condition. I hope it will work.
Good Luck
Are you cooling your sample properly during the sonication?
The sonication process produces a lot of heat; the cavitation bubbles can reach temperatures of 5500 Kelvin - the same temperature as the surface of the sun. Your protein is perhaps heat denatured during the process.
Put your sample container into an ice-water bath. Use a container that has a high surface to volume ratio to help with the heat dissipation.
Make sure the temperature of your sample never exceeds 40C.
I don't like sonication for cell lysis, be it bacteria, yeast or mammal. I had too many problems. For bacteria a french press works just fine. For small amounts, treatment with lysozyme and DNAse also works fine, followed by the needle as suggested by Abdalla Abdrabou.
No, I don´t think so. Sonication should not affect your results regarding purification.
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