How to Purify A Protein

[kiki06]

How can I purify a protein of interest? Which type of column should I use? I am trying to purify it for enzymatic assays.

thanku

[wexfrdo]

Does your lab/hospital have HPLC access? you can put your crude recombinant protein through an HPLC machine which has different columns.

Types of Columns I have used:

- anionic exchange
- cationic exchange
- hydrophilic column/hydrophobic column

-> that would help your purification immensely using HPLC....

[yiin]

Hi, I have purified a protein before but we knew its cDNA sequence. Thus we could easily expressed it with a GST tag and purify it after expressed in yeast.
You can use a plasmid that has a secretion tag that sends the protein to the media, and you don't have to kill or lyse the yeast, just keep them growing and collect media!

then with the GST fusion you can collect them on an affinity column and then cleave the GST tag away in case it is interfering with enzymatic activity....

try to express recombinant protein!

[MicroKiller]

I agree with the HPLC separation technique.. but beware for protein fragments... sometimes the HPLC separation may cut your protein to smaller fragments... if you are doing recombinant.. check your expression systems.. sometimes they have a his-tag which would allow you to use a Ni-Column to capture it via affinity separation.. good luck..

[aftabac]

hi
protein purification is a whole subject, and it is better to know the properties of the protein first, then according to the type of protein you choose the specific procedure for purfication. you must know the charge of your protein, pI of your protein and so many other properties.
first you must do i think some precipitation procedure, in order to narrow down the number of proteins, then you can purify manually by selecting the appropriate coloumn or by using HPLC or FPLC, but i depends on the protein.
and it is also important that whether your protein has a tag or present in fusion form or not, if it has some tag or present in some fusion form then you must purify it by affinity chromatography.
hope for the best
aftab
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[kiki06]

Thank you all for your great input.

I am trying a few things you all mentioned this month.


thank you

[krishan kumar]

how to purify opioid receptors

[MicroKiller]

opiod receptors?? u managed to isolate the u receptor from the brain/spinal cord?? i am not too sure of the receptor... but if u wnt to purify opioid receptors u can try affinity columns.. immobilize ur column with the opioid and pass ur receptor thru it... then it should be able to capture ur receptors..

[admin]

Yeah good idea microkiller... opioiod receptors... wow tough project...

[yongqing]

hello