Exer 10

Part A.

a.Failure to put bisacrylamide- the acrykamide would polymerize into long strands forming linear
polyacryklamide(an inert ethanol precipitation aid) and not a porous gel. Biscrylamide forms crosslink
between linear polymers forming a gel matrix. The amount of crosslinking and therefore the pore size
and consequent separation of properties of g el can be controlled nby varying the ratio of acrylamide to
bis acrylamide. (Shi and Jackowski 1998).

b. addition of excessive amounts of APS and TEMED- results in a decrease in the average poluymer chain
length , an increase in gel turbidity and decrease in gel elasticity.In addition, excessive amounts of
these iniatiators include oxidation of sample proteins (especially sulfhydryl-containing
compounds), increase buffer pH .reaction with proteins (Dirksen and Chrambach 1972;
Chrambach et al. 1976), and alteration of the banding pattern (Gelfi and Righetti 1981)

c. pH of gel buffers is equal to isoelectyric point of protyeins bbeing isolated- the solubility of the protein
is at minimum and is the point where protein accumulates to be collected.
https://info.gbiosciences.com/blog/bid/149959/what-is-the-role-of-the-isoelectric-point-of-a-protein-
in-its-purification

d. presence of bubbles in gel- causes the bands in the gel to become distorted as they will not form
consistently with each repetition and disrupt the physical medium of polyacrylamide. (Sun and
Anderson,2004).

e. electrophoretic run at 250 v- Higher voltage runs can result in gel distortion or compekte
disintegration https://www.sigmaaldrich.com/content/dam/sigma-
aldrich/docs/Sigma/General_Information/vol6_iss2_bionic_buffer.pdf

f. higher acrylamide concentration (ex 13%) for native page- As acrylamide concentration increases , gel
mobility decreases as migration is hindered by a larger particle size.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3785759/

Rationale of the ff:

a. Tracking dye- acts as a guide to allow the migration of the dye to be monitored.
b. Glycerol/glycerine in sample buffer- adds weight to the sampkle in which the sample sinks to the
well instead of diffusing out.
c. Running buffer- provides ions that conduct electricity from which the sample will migrate.
d. Cold conditions during the run-maintains the integrity of both the sample and gel
e. Spacer- determines the gel thickness

Part B.
Paragraph 1

Electrophoresis is the process of separating molecules c/oRozel

One of the widely used types of electrophoresis are NATIVE page and SDS Page. Native pages is used for
detrmining the purity and is based on net charge(more influemtial) and molecular wight in analyzing
proteins at their biological activity especially enzymes and antibodies. In this type, the proteins eluted
have high MW and are prepared in a non-reducing and non-denaturing buffer so the tertiary and
quartenary structures of the proteins were preserved which are applicable for in-gel detection of
fluorescent‐labeled proteins, for in‐gel catalytic activity assays, determining native mass and
oligomeric state of membrane proteins. On the other hand, SDS page used in dterming the
molecular weight & its number of subunits followed by purifying proteins prior to sequencing is based
solely on molecular weight. The proteins became negatively charged due to SDS and fully denatured to
linear polypeptides due to the presence of reducing and denatured agents hence, the relative MW of
proteins are low .This typeis applicable to for immunological and proteomic studies.

a.nature of conformation of protein being separated:

b. relative MW of protein bands obtained

c. basis of separataion

d.applications

Paragraph 2: There are two types of buffer system in used in PAGE namely discontinuous and
continuous system. A continuous buffer system, which exploits only one buffer in the gel, sample, and gel
chamber reservoirs, is most often used for nucleic acid analysis and rarely used for protein gel
electrophoresis. Proteins separated using a continuous buffer system use a concentrated sample and
tend to be diffuse and poorly resolved though, it is easier to perform. Conversely, the discontinuous
system which is mainly used in electrophoresis results in higher resolution of sample concentration. It
uses a dilute sample and utilizes a different gel buffer and running buffer.
https://www.thermofisher.com/hk/zt/home/life-science/protein-biology/protein-biology-learning-
center/protein-biology-resource-library/pierce-protein-methods/overview-electrophoresis.html The
two types of gels differ in ionic strength, pH and concentration called stacking gel (low concentration)
and separating gel (high concentration). The stacking gel contains sample wells and allows the
compression of molecules before separation. Meanhwile, separating gel based from its name separates
molecules based on MW (Caprette,2012). The major advantage these type is that relatively large
volumes of dilute protein samples can be applied to gels but good resolution can still be obtained.

Types of gels used

a. Buffer solutions used

b. Concentration of sample
c. Advantage and disadvantage

Paragraph 3: Two specific uses of PAGE

Page electrophoresis is applied in different fields of science namely, MW determination,
forensics, molecular biology, genetics and microbiology.Among these common applicationsof
PAGE ,a specific use of this type is found In a study of
http://www.microbiologyresearch.org/docserver/fulltext/ijsem/30/2/ijs-30-2-
460.pdf?expires=1523785034&id=id&accname=guest&checksum=6B6B4D3EAA0AB4EDDF9684F
572048043) that uses PAGE for differentiating arthrobacters from coryneforms on the basis of
their soluble fractions and assessment of efficacy of this method compared to the use of
chemical analyses of cellwall components of arthrobacters. Moreover, in the field of molecular
biology, two methods namely ,PAGE and neuron activation analysis were used for the purpose
of protein quantification.

Part C.
Paragraph 4: Methodology
Paragraph 5: AT time after running the PAGE, smiling or frowning effects can be seenfrom the
bands. The individual band smiling/frowning is often due to running of the buffer system at
ahigh voltage whereas a whole gel smiling/frowning is caused by overheating of the gel. The
cause of these defects is due to uneven application of current temp. throughout the set-up. The
current I solutions is conducted by buffer ions with small proportion being conducted by sample
ions. If the current is not evenly distributed to the gel, it will resul to the movement of the gel at
different speeds forming these shapes depending on the part of the gel the sample moves
faster. In addition, temp also affects the rate of migration and unevenly distribution of this
factor will also cause diiferent rates of movement of the gel.

Pargraopgh 6: Two other staining techniques to visualize protein

Besides using bromphenol blue, other staining methods can be used to visualize protein. The
use of silver staining deposits metallic silver onto the surface of the gel.It increases te level of
sensitivity approx. 100-fold. It can be used to increased the levels of detection in nucleic acids
and proteins. Furthermore, fluorescent staining uses ethidum bromide to react with nucleic
acids and proteins to yield a flurosecent addition product.

Part D.
Paragraph 7:
As seen in Figure 10.1 the first and second lanes from the left side and the last three lanes at the
right are filled with BSA extracts which showed that no bands separated from the sample
signifying thick bands thus considered pure. Moreover, the bean extracts showed a separation
of three bands indiciating that there were more than one kind of protein with different
molecules present. As serum proteins move towards the cathode, separated proteins appear as
distinct bands in the order of : first band- albumin; 2nd band lpha-globulins; 3rd band- beta 1
globulins 4th band- beta 2( Gallagher, ) Plants accumulate protein reserves in developing seed.
The major amount of these reserves constitute specific storage proteins like globulins (Shewry
and Casey,1999) which are the major syorgae proteins of mos cerelas. In this way, they are
proteins are protected against premature proteolytic attack. On the comparison of bean
proteins and BSA albumin is found both at the topmost portion because of it is the fastest and
lightest protein forming a large band and close to positive e-.Theoretically in a protein xtract,
bet glubins were the second bands to have thick bands followed by alpha glubilns and gamma
globulins. Errors might have occurred resulting to less visibility of the beta as it binds with
gamma globulis. Gamma globulins were the heaviest as they have the slowest mobility towards
the anode hence a light band was observed.