Dear Tilahun

there is no a un universal answer for your question, it depends a lot from the properties of your specific protein and for what do you need the protein sample and of course you need to distinguish the protein purification (preparative) from protein characterization (analitycs)

For example for research scale purification of soluble recombinant protein i'm a fun of N-terminal His Tag coupled with TEV cleavage site. This clone design in many case allow with 2 step IMAC purification which is simple, fast

You can find a video about it at the following link of my blod (proteoCool)

https://www.blogger.com/video.g?token=AD6v5dxhCeJUPx5UODBu4Y9hhJZix_l5JZcPb03ZUe7G3qL8GhoPUWZMm6z2wEJJED9OY8gn_ttNXrGNCdVurZ6zkB_oTSCrmRc90-ux7AQAOhaddCkXKREYmgNw87RSh58zQ4uVQVtz

however TEV cleavage require some specific buffer and condition that cannot work in all cases and is suitable for small scale research purpose (up to 100-200mg of recombinant protein) while is not so simple to scale up it in industrial scale because it is very expensive and not so reproducible in large scale, where generally the company, to avoid royalties and other issue, do not use TAG for affinity purification but are going with the expression and purification of untagged protein and in this case, more standard approaches are ionic exchange, ammoniun sulfate and or PEI precipitation become very usefull.

For analytics, i like a lot

SEC ( specially if coupled with MALS or LALA-RALS) that can provide you information about the aggregation state of your sample. Comparing the SEC profile of sample subjected to different stress conditions (eg froze thaw cicles, or incubation at different temperatures (eg 37°C, 55°C) you can also obtain some information about protein stability.

For rapid screen of protein thermal stability i like a lot the DSF (which is not a chromatographic approach), which is able with few ul of protein to give you an idea of the intrinsic thermal stability of your protein.

If you are interested you can find some more info about it in the following link

https://www.blogger.com/video.g?token=AD6v5dzr9KqZslTx9NeEjXx1XyPti8dU15eqYoE9TT8OEDaZQcNINuDKwzCAzVXkFWZA0btipKTr81doABh-VjpprwCcRukVcLhAXE_T5BzPxYbeQQA7VMFmc4DdpjS4XbOfxkwAhIWu

This are just some tips from my experience but the answer to your question should take a few hours to be exhaustive :-)

good luck

Manuele

Dear Tilahun

there is no a un universal answer for your question, it depends a lot from the properties of your specific protein and for what do you need the protein sample and of course you need to distinguish the protein purification (preparative) from protein characterization (analitycs)

For example for research scale purification of soluble recombinant protein i'm a fun of N-terminal His Tag coupled with TEV cleavage site. This clone design in many case allow with 2 step IMAC purification which is simple, fast

You can find a video about it at the following link of my blod (proteoCool)

https://www.blogger.com/video.g?token=AD6v5dxhCeJUPx5UODBu4Y9hhJZix_l5JZcPb03ZUe7G3qL8GhoPUWZMm6z2wEJJED9OY8gn_ttNXrGNCdVurZ6zkB_oTSCrmRc90-ux7AQAOhaddCkXKREYmgNw87RSh58zQ4uVQVtz

however TEV cleavage require some specific buffer and condition that cannot work in all cases and is suitable for small scale research purpose (up to 100-200mg of recombinant protein) while is not so simple to scale up it in industrial scale because it is very expensive and not so reproducible in large scale, where generally the company, to avoid royalties and other issue, do not use TAG for affinity purification but are going with the expression and purification of untagged protein and in this case, more standard approaches are ionic exchange, ammoniun sulfate and or PEI precipitation become very usefull.

For analytics, i like a lot

SEC ( specially if coupled with MALS or LALA-RALS) that can provide you information about the aggregation state of your sample. Comparing the SEC profile of sample subjected to different stress conditions (eg froze thaw cicles, or incubation at different temperatures (eg 37°C, 55°C) you can also obtain some information about protein stability.

For rapid screen of protein thermal stability i like a lot the DSF (which is not a chromatographic approach), which is able with few ul of protein to give you an idea of the intrinsic thermal stability of your protein.

If you are interested you can find some more info about it in the following link

https://www.blogger.com/video.g?token=AD6v5dzr9KqZslTx9NeEjXx1XyPti8dU15eqYoE9TT8OEDaZQcNINuDKwzCAzVXkFWZA0btipKTr81doABh-VjpprwCcRukVcLhAXE_T5BzPxYbeQQA7VMFmc4DdpjS4XbOfxkwAhIWu

This are just some tips from my experience but the answer to your question should take a few hours to be exhaustive :-)

good luck

Manuele

@ Manuele Martinelli thank you for nice answer! it is very helpful!. I need protein purification from bacteria!

Manuele Martinelli is correct, there are too many factors to consider to answer without narrowing down a number of variables. You mentioned bacteria as the source; so, recombinant expression or endogenous host protein? Which bacteria? Where is the protein expression localizing in the host (membrane, cytosol, periplasm)? What are the considerations for reducing conditions? What is the pI of the protein? What is the molecular weight of the protein? Is it monomeric or multimeric? If recombinant, does the protein have a purification tag? What is the downstream application and will purification conditions impact use? Is the protein stable to chaotropic buffers, high salt, extremes of pH? Are there cofactors which need to be preserved? These are the fundamental questions, and I am probably missing a few in this quick post.

Hi,

Great input Thomas and Manuele! Yes, there is a lot of factors that influence how to work. Personally I prefer IEX before precipitation as you have more control and possibilities to optimize in a controlled way. Contact me if you want to have more information about IEX chromatography, [email protected]

Dear, Tilahun Diriba Rabuma

Purification of protein takes place through the methods you have mentioned in your post.

But the question is what you will do to make your protein more purified? So, you should start with the most common method which is salting out followed with one or two methods from affinity chromatography, ion exchange chromatography or gel filtration chromatograph. This is mean that you can use more than one process of protein purification in consequent way to get your protein in a clear purified form.

However , high performance liquid chromatography is a technique used to separate, identify, and quantify each component in your mixture. It may not work for some samples. So you have to collect excellent information about the nature of your protein and put your alternatives of the purification methods to take a right decision of your work. I hope this comment will help you.

With my best wishes .

Mervat Mounir

@ Manuele Martinelli , Anna Heijbel @ Thomas J Esparza Thank you for your nice comments and suggestions. Actually, I am going purify amylase from bacteria. Thank you once again!

Try all three methods , size exclusion, ion exchange, ammonium sulphate precipitation . Use each method on a small scale for a quick determination of which is the best for scaling up.

For biological materials that may contain proteases it is good practice to include protease inhibitors.

If you have any information about the structure of the protein or proteins that you wish to purify then you may be able to select a method which is most appropriate for that specific purification . For instance if a protein is very acidic ( or basic) it may be best to use ion exchange as a first choice of method.