Proteins can be purified by size or by charge. Gel filtration chromatography on Sulfadex columns separates proteins by size. Ion exchange chromatography on DEAE- or CM-Sulfadex columns separates proteins by their charges. HPLC can also be used. SDS gel electrophoresis is not a separation method because SDS denatures proteins, and is used to check the purity of the final product.Can anyone explain me the procedures involved in isolation of proteins?
Column chromatography *can* be used to isolate proteins, as can SDS PAGE (polyacrylamide gel electrophoresis). These are both forms of chromatography.
Reverse-phase HPLC (High-pressure liquid chromatography) is a method of purifying proteins from a mixture of proteins (and possibly other substances) by their hydrophobicity. The principle is to use a column of packed beads which are hydrophobic in character. You dissolve your protein mixture in an aqueous solution (called the ';immobile phase) and pass it through the column. The proteins will stick to your column, as most proteins are quite hydrophobic.
You then gradually increase the amount of an organic solvent (like acetonitrile) you pass over the column (for example, maybe increasing from 0% to 100% over 1 hour 40 minutes). The different proteins will have different hydrophobic characters: in general, the longer a protein, the more hydrophobic, and the more hydrophobic amino acids it has in it, the more hydrophobic it is. As the percentage of organic solvent (called the ';mobile phase';) increases you will gradually reach a concentration which will start to remove the proteins from the column one at a time - the least hydrophobic will come off first, and the most hydrophobic will come off last.
At the end of the column, you have a detector, which will detect when proteins are coming off by UV absorbance (all proteins absorb UV at a wavelength of 230nm), so you can collect individual fractions which will hopefully contain only one protein.
After this, you can determine which proteins are in each fraction by other methods (like immuonoblotting, or mass spectrometry), and you will then (hopefully) have a pure sample of your protein-of-interest.
There are other methods of column chromatography - like size-exclusion columns, where the largest proteins come through fastest, and the smallest last.
Another refinement uses antibodies against your protein-of-interest, immobilised in the column. You pass your mixture over the column, and only your protein will stick to the antibodies. After running everything through, you wash with a concentrated salt solution, which removes your protein and provides you with a purified sample.
SDS PAGE is another method of separating proteins from a mixture by their size. You dissolve your proteins in a solution containing sodium dodecyl sulphate (SDS) - a detergent which gives the proteins a net negative charge. You then load your mixture into a well at one end of a gel of polyacrylamide.
A current is applied to the gel, so the charged proteins will migrate through the gel; the largest proteins move slowest (as they take longest to pass through the pores in the gel), and the smallest move fastest.
You will usually include a charged dye, which will migrate with the fastest proteins, and when this dye has reached the end of the gel, you stop the current and stain the gel with a protein-detecting stain (like coomassie blue) to show up where all the protein bands are. By comparing with a lane in the gel that contained a set of known-mass standards, you can estimate what weight each of the bands are.
SDS PAGE is not usually used to actually *purify* proteins, but to determine what is in each mixture, as the amounts loaded are usually rather small.
A similar method is used for isolating DNA from a mixture of different-length DNA strands.
A different method, called isoelectric focussing, involves not adding the SDS, loading your samples in the *middle*, and applying an alternating current to the gel (running first one way, then the other). This has the effect of concentrating each protein at a specific location, dependent on its *charge*, rather than its weight.
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