I am doing purification of a protein based on cyanogen glycoside contant from a plant. Can any one suggest me how can i determine its isoelectric point as it has not been fully characterized?
Hi If you know protein sequence you can predict pI using this online tool :
ExPASy- compute/Mw
Hope it helps!!
Do you know nucleotide sequence? try translating it into protein sequence online and predict pI??
Hey Gopal, Thanks. I know its easy to translate the sequence but as i mentioned it is not characterized. I didnt have cloned yet. I am extracting protein directly from leaves and i want to purify further so for that i want to know its pI.
Ok!! In that case you can try doing isoelectric focusing to know the pI of your protein.
It will separate protein based on your pI.
IEF Markers are also available which help to predict pI the way we look for MW of proteins.
I agree with Gopal and Jai!! First you need to purify your protein sample also knowing it molecular weight will be helpful in identifying your protein from you samples!!
If you know your molecular weight then you can run a 2D-PAGE to know its pI
Hope this helps!!
One needs to do exploratory scouting tests using various ion-exchange columns and different pH's to determine it's behavior. Many, many times the knowledge of the proteins pI is NOT a reliable indicator of how it will behave on ion-exchange media... it all depends on how the groupings of charges on the surface of the protein are arranged. So, it is much better to just go ahead and determine how it behaves. Read the ion-exchange section in Scopes, "Protein Purification: Principles and Practice" for examples of how to quickly scout out ion-exchange behavior.
Thank you so much Dean Cuebas., Jai Ghosh, Rohit Singh and Gopal Tiwari. :-)
for that purpose you need to purify your protein, then perform 2D SDS/DIGE... that involves iso-electric focusing (you may stop here too but for full characterization follow down). 2D SDS will separate your protein from others separately in form of spots. excise these spots and via LCMS route you may characterize your protein..
I wonder if you have purified the protein already? or it is still in the extracts? Have you got any information about your protein at all? e.g. the predicted size of your protein?
In case you do know the size, run a SDS-PAGE and send sample away for Mass-spec. By then, you should have your a.a. sequence. It should be straight forward from there.
something I have done over decades ago for my thesis: make use of ion exchanger resins. I prepared the protein in a buffer of low concentration (10-20 mM), the pH is not that important. Then, I added 100 mikroliter of the protein solution into several tubes that each containing 250 mikroliter anion exchanger resin (DEAE) equilibrated with about 50-100 mM buffer at pH values of 4.5, 5.0, 5.5, ... (increment 0.5 unit) ..., 7.0, 7.5. I did the same with cation exchanger (CM). Just incubate for about 1-2 hours (did mixing by inverting the tube every 10 minutes or so), and then spin down with simple bench mini centrifuge. I took the supernatant and check for protein and activity (since it was an enzyme). I checked on SDS PAGE and the protein bands are being corresponded with the activity assay. At least I knew by then the relation between which band represent my enzyme activity. Further, I plot the activity (y-axis) to the pH values (x-axis) of samples recovered from each resin. It may not give me the precise last digit of my protein pI but certainly give a hint on what pI it is about. Sorry for old fashioned way but I hope it helps.
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