Protein Purification: Methods and Techniques

Running head: PROTEIN PURIFICATION 1
Protein purification
Name
Institution

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Introduction
Protein purification is one of the most exploited research in the recent past. Albumin
purification, for example, is a very essential especial for patients having anesthesia or are under
very critical care conditions ( Schopfer, Lockridge, David, & Hinrichs, 2019). Purification process,
however, is not an easy task though. A lot of time and very expensive equipment are needed for
the success of the process. Ion exchange chromatography is one of the most preferred methods of
purification of protein (Schopfer, Lockridge, David, & Hinrichs, 2019). Ion exchange chromatography
has an advantage over other chromatographic methods of analysis in that it has high-resolution
power i.e. the ability to distinguish and separate very close but different protein molecules, hence
it is preferred for use in the purification process.
Question2: Method for the purification of the protein
Carboxymethyl cellulose exchange resin and Diethylaminoethyl exchange resin were
the two methods used in this purification process and their results compared using the DS-page
technique was used to ascertain the purity of the protein as well as the method that was of more
benefit in the purification process. Both carboxymethyl cellulose exchange resin and
Dithylaminoethyl exchange resin were filled in the ion exchange column as the packaging
materials instead of silica gel.
Diethylaminoethyl cellulose exchange resin with a relative molecular weight of about
90000 and particle size of about 60micrometre together with the sodium carboxymethyl cellulose
exchange resin having an average molecular weight of about 70000g and a particle size of about
35 micrometers was used for the purification (Schopfer, Lockridge, David, & Hinrichs, 2019).All
were prepared by following the standards laid down by the standard preparation protocol.
Preparation of the buffer solution and the pH separation requirement

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In the purification process for the protein, the buffer should always have a pH of 1 unit
that differs from that of the product. In the result obtained from the graph, the pH of the protein
was found to ranging between 7.6-9.5 this, therefore, means that the pH of the Diethylaminoethyl
exchange resin should be just above 10.00 and that of the CM-cellulose exchange resin just
below 6.7.The CM-cellulose resin forms an insoluble precipitate with the protein at a pH around
6.7.DEAE was prepared by adding 20 molar concentration of the tris buffer followed by chloride
ions as the counter –ions at a pH of about 7.6. CM-cellulose exchange resin, on the other hand,
was prepared by addition of 5o molar concentration of the lactic acid followed by sodium ions as
the counter ions (Schopfer, Lockridge, David, & Hinrichs, 2019).
The counter ion concentration was then determined for the DEAE cellulose exchange
resins first by the creating five different concentration 0of the tris buffer from the centration of
the counter ions ranging from 0.055 to 0.256 molar.1.5 milliliter of each of the created buffers
were added to an equal amount of the DEAE cellulose exchange resin.1.5 of the complex sample
of the protein was then added (Schopfer, Lockridge, David, & Hinrichs, 2019).The resulting mixtures
were then shaken by a shaker. A shaker is an instrument used in ion exchange chromatography to
shake a sample under analysis to create suspensions (Schopfer, Lockridge, David, & Hinrichs,
2019).The suspensions created through shaking are then collected out using a centrifuge. An
instrument for removing suspensions.
1.0ml of the resulting resolution from the shaker was then added to 1.5 ml of the washing
buffer shaking it thoroughly using the shaker for about 4 minutes and again centrifuged
(Schopfer, Lockridge, David, & Hinrichs, 2019). The shaking and centrifugation of the resulting
solutions were repeated severally until the best sample composition was achieved (Schopfer,
Lockridge, David, & Hinrichs, 2019).All the resins attached to the protein was then removed and

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separated out by adding 0.5 ml of the 1M tris buffer and then shaking the resulting solution for
about 4minutes, The resulting solution is then centrifuged for 5minutes (Schopfer, Lockridge,
David, & Hinrichs, 2019). The process was then repeated three times.The same was also done for
the CM-cellulose exchange resin using it is on buffer solution of 50 molar concentration and the
sodium ion concentration as the counter ions.
In finding out the concentration of the human serum albumin detached from the resin
(Preto, Matos-Filipe, Koukos, Renault, Sousa, & Moreira, 2019). Suspension production and wash out
washout was done to the CM-cellulose exchange resin by addition of 0.05 molar concentration
for the counter ion and 50mM of the formic acid to produce 0.03, 0.5. 0.15 And 0.2 molar
concentration (Preto, Matos-Filipe, Koukos, Renault, Sousa, & Moreira, 2019).It is worth noting that
0.1 molar concentration of the chloride ions was used as a starter for the DEAE cellulose
exchange resin while for the CM-cellulose exchange resin, 0.05 molar concentration of the
sodium ions were used in order to remove the human serum albumin.
The protein purification technique was achieved by washing the resin with 3v fold of the
primary buffer in which each vial was filled with 0.4 ml of the resin (Preto, Matos-Filipe, Koukos,
Renault, Sousa, & Moreira, 2019) Initially 0.4 ml of the already prepared tris buffer with 0.1M
concentration of the chloride ions were added to the resin and shaken for 5minute to create
suspension (Preto, Matos-Filipe, Koukos, Renault, Sousa, & Moreira, 2019). The resulting mixture was
then transferred to the centrifuge for collection of the suspensions crested through shaking (Preto,
Matos-Filipe, Koukos, Renault, Sousa, & Moreira, 2019). 0.5 ml of both the plasma protein and the
starting buffer was then added to the resin and shaken again with the shaker for 5 minutes and
then centrifuged again for 5 minutes to collect out the suspensions.0.5 ml of the solution was
obtained and the rest wasted ( Preto, Matos-Filipe, Koukos, Renault, Sousa, & Moreira,2019) .0.6 of

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the starting buffer was again added to the resulting 0.5 ml mixed with the shaker and then
centrifuged to collect out the sample suspension.0.3 ml of the final solution was then added to
the starting buffer contained in the vial number 1 (Preto, Matos-Filipe, Koukos, Renault, Sousa, &
Moreira,2019). The resulting solution was then mixed with the shaker, shaken to create suspension
and then centrifuged to collect out as suspensions.
0.2 ml of the latter solution was then added to 0.4 of the washing buffer then again
shaken for 4minutes using the shaker followed by centrifugation by the centrifuged to collect out
the suspensions (Preto, Matos-Filipe, Koukos, Renault, Sousa, & Moreira,2019). The shaking and
centrifugation of the resulting sample for this sages were repeated twice. All the resins attached
to the protein were then separated out by adding 0.4 ml of the 1Ml tris buffer to the resin mixed
with the shaker for 2 minutes and then centrifuged to collect out the suspensions.
Question3; Technique for the separation of the protein
The .SDS-page technique was then used to analyze the resulting sample. As shown in
figure 1.1 below

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Figure 1.1
Figure 1.1 showing the result of analyzing albumin analyte on an SDS-PAGE when
DEAE cellulose exchange resin was used as the stationary phase.
The SDs page technique used for the analysis of the sample solution gave the above
result on a DEAE cellulose exchange resin (Ma, Liu, Chen, Lin, Zheng, Miao, & Xie, 2019). The
following analysis was made.Lane on the SDS page above shows the protein marker. Lane
2,3and 4 shows the protein extracted from sample first, second, third and fourth addition of the
samples into the column. Lane 5 6 and 7 however representing the washed proteins. (Ma, Liu,
Chen, Lin, Zheng, Miao, & Xie, 2019).The proteins washed during the three stages of the washing

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process. Lane 8 to 10 represent protein retrieval from the column (Ma, Liu, Chen, Lin, Zheng, Miao,
& Xie, 2019). They show how the extracted proteins from the three stages were retrieved. Cm
cellulose resin, on the other hand, gave a perfect result as compared to the one obtained by the
DEAE cellulose exchange resin as shown in figure 2.1 below.
Figure 2.1; showing the expected results of analysis when protein sample was analyzed using the
SDS-PAGE when CM-cellulose exchange resin was used as the stationary phase as extracted
from (Ma, Liu, Chen, Lin, Zheng, Miao, & Xie, 2019).
SDS page analysis of the protein samples on a CM-cellulose exchange resin.In this
analyzed result, lane 2 represents the protein maker.Lane 1, 3, and 4 represent the proteins
extracted from the three stages of the sample addition (Ma, Liu, Chen, Lin, Zheng, Miao, & Xie,
2019). Lane 5, 6 and 7 on the SDs page above show the washed proteins from the three stages of
protein washing process (Ma, Liu, Chen, Lin, Zheng, Miao, & Xie, 2019). Lane 8S, 9 and 10,
however, represent the protein extracted from the stages of the protein extraction process.
Question3; Technique for the separation

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From the observations made on the SDS page sample analyzer on the DEAE cellulose
exchange resin for the figure 1.1 and on the CM-cellulose exchange resin for the figure 2.1
above, all the tests were performed with the start buffer (Ma, Liu, Chen, Lin, Zheng, Miao, & Xie,
2019). It was observed that the first column contained a larger amount of the extract of both the
albumin and other proteins which was attributed to the outdated version of the resin as well as its
low capacity (Ma, Liu, Chen, Lin, Zheng, Miao, & Xie, 2019). Three lanes from figure 1.1 relating to
the extraction phase were all from the DEAE cellulose resin indicating that albumin contributed
to over 60% of all the proteins purified.
This means that the purification process was 55% successful. However, at the third stage
which is the resin retrieval phase .almost three-quarters of the proteins were washed out and 60%
of the all the protein purified were albumin. For the CM-cellulose exchange resin in figure 2.1,
the three lanes related to the protein extraction showed that almost all the other protein other than
the albumin was washed out from the bands. Showing that the albumin had 70% of proteins in
this composition. For the third phase, the resin was then retrieved.From the result of the SDS
page analyzer, CM-cellulose exchange resin yielded better result as compared to the DEAE
cellulose exchange resin.
Question 1: Determination of molecular weight and the
isoelectric point for the unknown protein
Determination of the molecular weight of a protein molecule using an SDS-PAGE
involves the comparing of the standard values of the molecular weight obtained from the SDS –
PAGE analyzer and determining the estimated value for the unknown protein (Ma, Liu, Chen, Lin,
Zheng, Miao, & Xie, 2019). It can also determine graphically and using the general equation of a