Virtual Lab: Protein

Purification
JV Gamo | CH152 - B

The process of protein purification may be divided into three main parts: capture, intermediate
purification, and polishing. (MCB Harvard University). In the capture stage, the target protein is
isolated and concentrated. The process of 2D page and immunoblotting is part of this stage. Next is
the intermediate purification stage, where most of the impurities, such as nucleic acid and other
proteins, are eliminated. The process of gel filtration is part of this stage. Lastly, the polishing
stage is where the rest of the remaining impurities are discarded. The process of assaying enzyme
activity and subsequently pooling fractions of the sample based on the assay, are included in this
final stage.
Seen here in the image above, the enrichment value is a mere 1.0, by the end of the protein
purification process, the enrichment level will have been raised by a significant amount.

The first process done is the two-dimensional gel electrophoresis or 2D page,

which is commonly used for proteins. Herein, protein mixtures are separated
by two properties in two dimensions on 2-D gels. The two properties
identified isoelectric point in the above example are isoelectric point (x-axis)
and protein mass (y-axis).

The above picture shows immunoblotting, which is a technique used to

analyze or identify proteins in a mixture. It involves separation by
electrophoresis and subsequent staining with antibodies to isolate the protein
in question. This shows at which isoelectric point value and protein mass
value the target protein is located (pH 8.0 and 55,000 Mr, respectively)

Now, we have the task to choose the most appropriate gel matrix to use for
the gel filtration. Ultragel AcA 34 has the only appropriate fractionation
range of 20,000 400,000 molecular weight, which included 55, 000, the MW
of our protein, within its range.

In the graph shown above, the red portion is the only fraction of the sample
wherein enzymatic activity is observed. This observation is possible due to
the enzymatic assay procedure, which is for the purpose of measuring
enzyme activity.
Portions 75-90 were then subsequently pooled and isolated because they
were the only fractions that exhibited enzyme activity.

After, the gel filtration procedure, the enrichment of the protein is now up to
4.7, from 1.0. This means that some of the impurities have now been
eliminated. But lets see if we can do another procedure to eliminate the rest
of the impurities.

Here is where another separation process

called
ion exchange
chromatography comes in. Separation through this process is based upon
the reversible adsorption of charged molecules to the inactive ion exchange
groups of the opposite charge. (MCB Harvard University). Hereafter, the some
or most, if not all, of the remaining impurities can be discarded.

In the graph shown above, the red portion, again, is the only fraction of the
sample wherein enzymatic activity is observed. This observation is possible
due to the enzymatic assay procedure.
The fractions within the red curve (i.e. 60-120), are then spooled and isolated
because, again, they were the only fractions that exhibited enzyme activity.

After the ion exchange chromatography process, the enrichment value is now
up to 51.1 from 4.7, which means more impurities have been eliminated after
this step. There is no particular indicator as to how purified the protein has
been, but the increase of the enrichment level is a telltale sign of purification,
per se.

SOURCES
http://www.agbooth.com/pp_tut_onlin
e/Protein4.html
http://labs.mcb.harvard.edu/Gaudet/
Resources_Files/GEHealthcare_chrom
atography/Don't
%20move/18113229AB.pdf