We tried with amm. sulphate 100% saturated solution (4.1 M solution ). First tried with equal amount of both solutions(1: 1) followed by 1:3 proportion. Even then there was no precipitation. We need to precipitate plant proteins. Can anyone suggest a better method for this problem?
Did you quantitate the protein in your sample first? It is crucial to make sure your extraction yields enough protein to even be useful to work with. Depending on what you need it for, you may need a lot of more protein. It's difficult to move on to precipitation until you have verified you have enough material initially.
What method did you use to extract the protein? Also, the choice of method of precipitation highly depends on how you need the protein after precipitation - do you need it in a denatured form or not? How much protein you have extracted is also important before you choose a precipitation method. You need to determine the quantity extracted then you can choose a precipitation method depending on the sensitivity of the method, which is guided by the quantity extracted. Be sure to store your crude extract safely and in a good buffer to avoid degradation of your protein or you extract and precipitate immediately then proceed to separation and sequencing.
Thanks for the suggestions.How can we quantify the unknown proteins in the plants
We need the functional proteins after precipitation.So which will be the best method?
We have the crude extract from the plant in powder form.So from that we are trying to isolate the protein of interest which is an unknown protein.what we thought is that when we do the precipitation ,all the proteins in the plant will be precipitated including this unknown one also.What we did is certain amount of powder was dissolved in known quantity of distilled water.After that equal volume of saturated solution of amm.sulphate (4.1 M = 100% saturation) to it.We need this unknown protein ,considered to play a very crucial in detoxifying certain toxic plant principles in active form to do our experiments.
From this, i see you need the protein in its native form, which calls for caution to avoid denaturing it since you have an interest in its biological activity. Then you need to employ protein purification methods first before identification. Note that you cannot identify your protein of interest while its in a mixture! I suggest you quantify the extract to determine the amount of crude protein, then start your purification, which will be assay guided. You can use HPLC if you have a good column or you decide to go the long way, ion exchange, column chromatography, etc. Be careful, purification of proteins required in their native forms can be tricky. Temperatures play a crucial role, a slight mistake may render your protein inactive, meaning you start afresh from extraction. After the first separation and assay, you can run a gel to get a clue about some characteristics of the protein i.e how close it is to the rest of the proteins so that you can design your next step from a point of knowledge.
Wish you well.
I use the Pierce BCA kit for quantification, but Lowry can be done with common lab chemicals and a spectrophotmeter. You can google that info. I believe ammonium sulfate will precipitate proteins without denaturing them, but you will need to read up a little more about it. Do NOT use any detergents. When you get an acceptable yield, you will need an control to prove that the protein is still fuctional. You can precipitate a commercially availabe (and cheap) enzyme with known properties and see if it remains active to serve as as a control.
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