The proteins need to be enzymatically active, planning to use Taq. Polymerase for PCR reactions. I am looking to create a protein extract that can be used for diagnostic PCR. So the protein has to be able to polymerise DNA, but the DNA it produces won't be used downstream.
I would propose to use ultrasound for cell disruption to obtain a lysate containing proteins and DNA
Fractional precipitation with ammonium sulphate will remove a lot of protein and is cheap and easy and quite reproducible and will concentrate your protein.
You need a native purification method. As you are working with a soluble protein it suggest that you add a poly-His tag to your protein of interest and a purification resin or magbead that can be regenerated.
Therefore you can use it multiple times that should reduce costs in the long run.
For maximum purity of would go with Co-NTA resins / Magnetic beads. They might give a lower protein yield but the result is much purer:
https://cube-biotech.com/products/protein-purification-products/his-affinity-resins-magbeads/co-nta/
If you desire a higher protein yield, then INDIGO-Ni resin / magnetic beads should be your best option.
https://cube-biotech.com/products/protein-purification-products/his-affinity-resins-magbeads/indigo-ni/
Just remember to follow the native purification protocols.
as you mentioned that you need to use it in diagnostics. You cannot use any simple method to isolate your protein. You need to purificate your protein by affinity Chromatography. Tag based purification (his tag etc.) followed by a second step ion exchange can be used.
Thanks to Stjepan for his comment.
However, a sonic bath should be used for cleaning of items (e.g. of glasses) not for cell sonification.
Dear Nico
You protein is a TAQ? IS thermostable?
Because, generally considering that TAQ are stable at high temepratures the simple approach is perform the cell lysate with sonication or french press, remove the most of cell membrane with low speed centrifugation, incubate the unclear surnatant at 60°C for 30 minutes (which is able to denature and induce precipitation of the most of other proteins), perform high speed centrifugation and recover your purified TAQ in the surnatant.
best regards
Manuele
Manuele Martinelli I have never heard of that method before! I'm going to try it! Are there other methods of lysis that will work for protein recovery that you know of? I don't have a sonicator at my lab. Grazie mille!
Dear Nico
If you do not have sonicator or french press, You can try the cell litic express (Sigma aldrich c1990 ) which is a non denaturing lisys agent containing chaps and lisozime.. it is More expensive that standard buffer used for sonication therefore in general i like it for screening and lysis in parallel of many small biomasses. But in your case if you not have any sonicator, it can be an alternative expecially of the expression level is high and you do not have to lisate high biomasses.
1 bottle could be dissolved in 25 ml of milliq water and used to lisate up to 1.5 gram of e.coli wet biomass. To perform the lysis: Resuspend the pellet with the cell litic solution and incubate 30 minutes at room temperatire under agitation ( shaking or Stirring)
Good luck
MANUELE
Nico Crudele Please go through these articles :
Article A Simple and Rapid Method for Preparing a Cell-Free Bacteria...
Article A simple, inexpensive method for preparing cell lysates suit...
Article A simple, inexpensive method for preparing cell lysates suit...
If you are purifying Taq for genotyping PCR and have a pTaq plasmid or equivalent, there are a number of old sources for purification that work well. I have used these to make Taq about every 5 years and it works for most tail biopsy PCR reactions. You have to titer the activity to make sure you have a good stock.
https://media.addgene.org/data/27/29/165595e0-af64-11e0-90fe-003048dd6500.pdf
Article A simple and efficient method for extraction of Taq DNA polymerase
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