Hi, all :D
I'm doing short protein expression (~17 kDa, pI 9.6) from Bl21de3 with c-term His tag.
I purify it by IMAC (or affintiy chromatography).
When I do SDS-PAGE and WB with Anti-His Ab, I got a predicted target band (but it looks near 18kDa) and other SDS band.
I found it was cleavaged with Western blot near N-terminal.
I think it was cleavaged when lysis or purification.
And when I store the eluate at -20degreeC and melt it again, or keep it at 4 degreeC), then the target band is disappeared.
The suspension buffer is 50mM Na-phosphate/300mM Nacl (pH 8.0) and eluted with 300 mM imidazole in suspension buffer.
I think there is a fragmentation was happened in the solution.
I cannot sure if I have to use protease inhibitor or EDTA (how much of the concentration?).
In the case of EDTA, it is will strip off the Ni from NTA. So, I'm not sure how much of EDTA is suitable. My column is possible until 1 or 2 mM, I heard.
Is there anyone who suffering the problem like me?
Thanks :D
Dear Soy
Regarding protease inibition you can:
1) Add ad EDTA Free protease inhibitor cocktail before cell lysis
https://www.thermofisher.com/order/catalog/product/A32965
Or
https://www.thermofisher.com/order/catalog/product/A32965
Whose are very simple to use.. giù can just add a tablet in the lisys buffer just before purification.
One poter possibility, if you are that the éluted protein is quite good and the degradation happen after Storage is and the edta in the sample after solution from IMAC and if is possibile perform a second purification step ( eg SEC ) asap to remove the possibile proteases still present on the sample.
It you are not able to present degradation in this way, you need to tra other solution as:
Change e.coli strain or expression stategy (periplasmic expression );or host ( for example try a gram positive eg brevibacillus)
Change expression condiction (eg infection at Lower temperature)
Send the degraded band to n term edman degradation and sequencing to understand the protein digestion site and stabilize it by mutagenesis.
Each protein has is own properties and story. There is no a Universal solution this make the protein fiale challenging but very intriguing.
Good luck
Manuele
What Manuele wrote is really good advice.
But there is another option: It is true that Ni-NTA beads (or any NTA or IDA based IMAC ligands) are stripped by EDTA.
However there is a third option: INDIGO-Ni
https://cube-biotech.com/products/protein-purification-products/his-affinity-resins-magbeads/indigo-ni/
It withstands EDTA concentration of up to 20 mM while still functioning the same way as Ni-NTA for His Tag protein purification. Maybe this can help you too.
You can use EDTA even though you are doing an IMAC, as long as its concentrations are not too high, try with 0,2 mM or 0,5 mM, it works fine.
Otherwise, you must use some antiproteases, there are already a lot of available antiprotease cocktails available if you do not have your own.
Add them as soon as you resuspend your bacterial pellet after culture, and keep them until the purification steps, or even during purification steps, until you are sure that there are no more proteases in the solution.
Keep your protein on ice or at 4°C during the whole time.
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