BIOM3003 distributions in the covalent and ionic bonds

BIOM3003 REPORT- Part A

 

Introduction:

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Serum Protein Electrophoresis
(SPE) is a laboratory technique which is used to separate proteins in the blood
according to their size and charge. It is an easy and inexpensive way to
diagnose patients who are suspected to have multiple myeloma and other diseases
linked to serum protein. Serum is the liquid portion of the blood which does
not contain any clotting proteins but contains other proteins like hormones,
antigens and antibodies. The serum can be obtained by centrifuging the blood so
that the cellular components are removed.

 

In SPE, diluted samples of serum
are pipetted into a gel which is exposed to an electric current which expose the
major fractions present in the serum protein which are serum albumin, alpha-1
globulins, alpha-2 globulins, gamma globulins and beta globulins. In this
procedure, the proteins will be separated according to their electrical
charges. The electronic distributions in the covalent and ionic bonds found in
the proteins determine their electrical charges at specific pH. When the
samples are placed in the solution, the ionic species migrate towards the
cathode or anode according to their charge.  

Only a small fraction of the
total serum protein makes up the Gamma globulins which are essential for the
diagnostics of gammopathy.

Aim: To interpret the SPE to
identify the unknown constituent in the test sample.

 

Materials:

 

Materials needed for serum
samples-

-4 tubes

-Undiluted Standard serum (sigma
H4522)

-Purified serum test sample

 

Materials needed for electrophoresis:

-Helena SAS-MX SP kit (Helena
laboratories; catalogue number: 100100)

-Acid blue stain, Destain,
Methanol to fix the gel

-Purified water to make the
buffers

-Gel Tank

-Power supply

-Dryer

-Shacker

 

Methods:

To prepare the dilutions, 4 tubes were taken
where tube 1 is empty and the other tubes contain 10µl of purified water. 20µl of undiluted neat serum is pipetted into tube
1 and 10µl of distilled water was pipetted into the other three tubes. 10µl of
tube 1 was transferred into tube 2 and mixed well. 10µl of tube 2 was
transferred into tube 3 and so on until Tube 4 has been diluted to a ratio of
1:8. Two sets of these dilutions were prepared.

The cellulose acetate gel
was removed from its packaging and placed onto a paper towel. The overlay on
the gel is removed and the blotter C was used to blot the surface of the gel
and then discarded.  The sample
application template was aligned at the edge of the gel with the arrows found
on the template. Blotter A was then placed carefully onto the template and a
finger is rubbed across the slits to make sure there is good contact.

3 µl of undiluted and diluted
samples were carefully pipetted into each slit in the cellulose acetate gel
where the first 4 slits were pipetted with standard sample dilutions and the
last4 slits were pipetted with purified serum components. The samples were allowed
to absorb for 4 minutes and blotter A was used again to remove any excess
sample. 25ml of Tris-Barbital buffer was poured into the inner section of the
SAS-MX chamber and the gel was positioned, with the agarose side down, into the
chamber by aligning the negative and positive sides with their corresponding
positions. The gel was electrophoresed for 30 minutes at 80 Volts. The gel was
then fixed in methanol for 5 minutes and then left to dry at 60-70°C.

The dry gel was then
immersed in a stain solution for 10 minutes and shortly after it was destained
in destain solution in 2 x 1 minute washes until the background of the gel was
clear from stain. Finally, the gel was briefly washed in purified water and
dried.

Results;

 

Figure 1 shows the results from
the experiment. Lanes 1-4 show the migration of standard serum samples and
Lanes 6-9 shows the migration of the test samples from the cathode to anode. In
the standard serum samples, the darkest and fastest moving band that can be
seen is the Albumin as it is the most prominent protein in the serum. The
broadest band is Gamma globulin where some migration towards the negative electrode
is seen. The migration positions of gamma globulin are the same in the standard
samples and the test samples. The migration in the test samples can only be
seen in the gamma globulins region with no migration towards the anode.

 

 

Discussion:

 

Albumin is the most abundant globular protein found in the
serum which can be seen in Figure 1 as the band is very dark and thick. It also
has a very negative charge which means that it travels farther than the other
serum proteins towards the positively charged anode.

The
beta globulin band is darker and more prominent than the alpha 1 globulin and
alpha 2 globulin bands. This could be due to the beta globulin having more protein
constituents than alpha globulins. Some protein components of beta globulins
include ?2-macro-globulin,
fibronectin, transferrin and ?-lipoprotein. It can also be observed that alpha
1 and alpha 2 have separate bands which are less intense. In comparison, beta 1
and beta 2 globulins come under the same band which means that there is a
higher concentration of Beta globulins. Serum
proteins are negatively charged which explains that the samples travel towards the
positively charged anode. However, some of the gamma globulins migrate towards
the cathode away from the slit as
seen in Figure 1. This can be due to the buffer moving towards the cathode and
the weaker negatively charged gamma globulin molecules are not able to overcome
this force so migrate with the buffer. Lanes 6-9 show prominent, broad and
diffused bands in the gamma globin region when compared to the standard serum
samples. The low migration of this band suggests that the region contains heavier
immunoglobulins which have a weaker negative charge and a lower pl which
explains the migration towards the cathode. The bands in Lanes 6-9 appear to be
dense which suggests either a monoclonal or polyclonal increase in production
of immunoglobulins. Polyclonal gammopathy is characterised by diffused and
broad bands which can be seen on the gel.

In the future, accuracy of pipetting samples into the slits
of the gel could be improved as the first test sample may have leaked and
contaminated the ½ slit affecting the dilution.

 

SPE is cheap and easy to prepare. The cellulose agar produces
sharper bands. A quicker and more efficient method that could be used is Capillary
Zone Electrophoresis which can offer approximately 90 samples per hour.

 

 Component B:

 

Monoclonal Gammopathy (MG) is a condition in which
there is an abnormal production of immunoglobulins from a clone of B cells causing
the production of Paraprotein, which is an abnormal protein, that accumulates
in the bone marrow. Diagnosing MG is very critical as chances are high that the
patient may develop myeloma. There are many ways in which MG can be detected
such as Serum Protein Electrophoresis, Capillary Zone Electrophoresis and Immunofixation
Electrophoresis (IFE).

 

IFE is the detection of monoclonal antibodies or
immunoglobulins in serum protein or in urine. The technique is similar
technique to Serum Protein Electrophoresis however, this technique is faster
and more sensitive. The principles of IFE are to detect isotopes of the monoclonal
gammopathy in the sample. A reaction specific antibodies with the gel allows
observation of monoclonal isotypes, if present in the sample.

 

The technique involves four stages:

-Gel electrophoresis is used to separate the
proteins according to their size along a gel against electrical current

-Specific Antisera with antibodies are laid on the
gel to bind to their corresponding antigen and form a complex to fixate them
onto the gel. If M-protein is present in the sample, a band will form.

-Unfixed complexes are removed from the gel by
washing with saline

-An acid violet stain is then used to stain the
protein present on the gel to do further analysis.