We have our protein from the crude microalgae from that we have to purify the proteins first then we would like to know what are the compounds are present in that whole sample so kindly share some suitable methods and give your valuable suggestion. If we do HPLC how I can prepare the sample? Please suggest some methods.
Are interested in high molecular weight proteins or low molecular weight peptides? Also, do you want to retain the native structure of proteins, or do you just want to know which ones are there? Please clarify.
If the genome sequence of the algae is known, mass spectrometry-based proteomics techniques on a trypsin-digested extract will be allow you to identify which proteins and peptides are present.
If you do not know the genome sequence, you can use mass-spec sequencing to learn the sequences of peptides that are separated by HPLC.
we need low molecular proteins and also we want to retain the structure too
A standard fractionation procedure for a crude extract intended to separate proteins while retaining native structure might go something like this (variations are possible, of course). All operations should be conducted in the cold, preferably 4oC.
The first fractionation step could be ammonium sulfate precipitation. Solid ammonium sulfate is added gradually to the extract with constant stirring to reach various stages of saturation. After each step is reached, the extract is centrifuged and the pellet is collected. The next addition of ammonium sulfate is made to the supernatant. Each pellet can be used as the starting material for chromatography.
The 2nd fractionation step could be gel filtration chromatography. The ammonium sulfate pellet is redissolved in a low-salt buffer in a small volume and the sample is passed through a gel filtration chromatography column. Fractions are collected as the column is eluted with the buffer. This separates the proteins based on their sizes, but may also be affected by their shapes.
Each of the fractions from the gel filtration column can be further fractionated by anion exchange chromatography or cation exchange chromatography. The sample is applied to the column and the column is eluted with a buffer containing a gradually increasing concentration of salt while fractions are collected.
This methodology is normally applied to the purification of a single protein. Using it to fractionate a crude extract will require a huge amount of chromatography and result in a very large number of samples, each of which will probably contain several proteins in various amounts. The protein content of each sample can be separated by size for analytical purposes using SDS-PAGE. Bands from the gel can be cut out and sequenced by mass spec for identification.
Since you are interested in low molecular weight proteins, you can reduce the amount of work by selecting only the later fractions from the gel filtration columns.
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