Mass spec and PAGE

SDS -PAGE is the commonly used method to check the purity of a protein. You can cut the single band and do sequence analysis to compare with your standards.

Thanks

SDS-PAGE

Dear colleague,

If the peak distribution is narrow and a Gaussian distribution profile is observed, they may validate purity but not be assumed as a confirmed criteria of purity. Deformed tails and non symmetrically distribution in elution profile denote protein contamination.

An easy method to test protein purity is not possible for publishing purpose for some journal but some procedures are generally accepted. Best low cost and accessible methods are electrophoretic procedures (SDS-PAGE or other PAGEs according to the specific criteria of discrimination) selecting the appropriate gel % and/or varying % and Gel filtration profile depending on bed size selection and protein molecular weight.

Protein purity test do not need necessarily standards, except for validating the right elution profile expected for a known molecular weight (MW) or for calculating approximately the MW for the protein of interest.

Helped you?

Is there any easy method to test protein purity?-- The answer to your question is very simple: it depends on what type of protein are you interested in (and purifying). The easiest case is when the protein has a chromophore and the spectrum of the protein is known. In this case the known molar extinction coefficient belonging to the absorption peak of the spectrum provide you some help in testing the purity of your fraction. Not having any chromophore in the protein in question you might follow and test some "specific enzyme activity" and determine the specific enzyme activity in the different fractions in the different purification steps. When you reach the literature value you can say you might have got the protein in question as pure as possible. Of course, in each case the SDS-PAGE + some staining (mostly the silver staining) would provide rather firm support for the purity. If you know NOTHING about the protein (enzyme) in question, you would need more expensive (sophisticated) techniques (like MS/MS) after the mentioned SDS-PAGE+some staining and cutting the band in question.

To all the good ad:vice already given I would only add a couple of points:

1. You need to know what you regard as sufficiently pure - 99%? 99.9%. If you are using SDS-PAGE this will determine what is an adequate protein loading. If you want to see a nice clear band for your protein you may use a moderate loading that does not show up the faint bands of low-level contaminants. If you really want to see the contaminants you need to 'overload' the gel so that the main band looks like an ugly spill! Also, as mentioned by Alajos, silver staining instead of Coomassie Blue will give greater sensitivity.

2. You need to think exactly WHY the protein needs a particular degree of purity. Some years ago we wanted to use antibody detection in order to clone a bacterial protein. We had a lot of wonderfully 'pure' protein - i.e. it looked brilliant on a highly-loaded SDS-PAGE gel. A colleague advised me to use a generous dose of protein to inject the rabbit in raising the antibody. By a twist of fate the gene we really wanted to clone turned out to be toxic to E-coli, but we succeeded in raising a large number of clones for all the non-existent impurities! The rabbit was more sensitive than our gels.

3. Are you only concerned about the absence of other proteins or are you also concerned about the state of your own protein? A single band on a gel could conceal 'pure' but denatured protein or pure protein depleted of an essential prosthetic group. Accordingly considerations of specific activity are very important too.

Analytical HPLC

Thanks to all protein scientists. I was really helped and now have clearer idea about the protein purity. I am not having a particular protein to purify at this moment, just reading papers and come up with this question.

a) SDS-PAGE is acceptable but might not be able to tell trace amount of impurities.

b) Spectrum method.

c) Analytical HPLC. I am pretty interested in this method since it is available in my lab. Will it be similar to small molecular compound like checking the UV absorption and peak symmetry? Can MS/MS tell protein purity?