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How to Purify A Protein

How to Purify A Protein - Protein Science

How to Purify A Protein - Post Questions and Discuss Protein Structure and Function, Protein Localization etc.. Please do not post Proteomic questions here, there is a Proteomic Forum below.


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  #1  
Old 02-27-2006, 07:24 AM
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Red face How to Purify A Protein



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
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Old 03-03-2006, 11:58 PM
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Lightbulb HPLC access?

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....
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Old 05-02-2006, 01:49 AM
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Default Re: How to Purify A Protein

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!
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Old 06-20-2006, 08:53 AM
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Default Re: How to Purify A Protein

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..
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Old 06-20-2006, 11:32 AM
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Default Re: How to Purify A Protein

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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Old 06-20-2006, 05:01 PM
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Default Re: How to Purify A Protein

Thank you all for your great input.

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


thank you
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Old 08-14-2006, 05:05 PM
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Default Re: How to Purify A Protein

how to purify opioid receptors
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Old 08-17-2006, 09:48 AM
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Default Re: How to Purify A Protein

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..
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Old 08-17-2006, 08:04 PM
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Default Re: How to Purify A Protein

Yeah good idea microkiller... opioiod receptors... wow tough project...
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Old 12-23-2006, 03:46 PM
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Default Re: How to Purify A Protein

hello
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