1. Study of Reduction of Disulfide Linkages in Aggregated Bovine Gamma Globulin Using
SDS PAGE
Winona State University
Carissa Schieffer
Research Advisor: Dr. Myoung Lee
March 21, 2016
Statement of research problem
Amyloid fibrils are protein polymers that the body produces normally. These amyloid fibrils are
insoluble causing problems in the cell.
Image 1: Three dimensional models of amyloid fibrils from different sources. (a) View down the
long axis of the fibril (Tycko, 2002). (b) Shows the direction that the amyloid fibrils is formed
(Ritter, Maddelein, Siemer, Luhrs, Ernst, et al. 2005). (c) Atomic structure of the microcrystals
assembled from the GNNQQNY peptide (Nelson, Sawaya, Balbirnie, Madsen, Riekel, et al.
2005).
The structure in image 1 is a long chain of polypeptides stacked on top of each other forming
amyloid fibrils. For example, you could think of a big stack of paper piled on top of each other.
However this structure is insoluble unlike native proteins that are soluble. When this happens
inside or outside of neurons, the aggregated protein blocks off and restrains neurons. If these
neurons are blocked off this can lead to diseases such as, Parkinson’s and Alzheimer’s disease.
Type 2 diabetes is also characterized by amyloid fibrils.
Bovine Serum Albumin (BSA) and gamma globulin are both proteins derived from cows.
Gamma globulin are antibodies which are very important in protecting the organism from
diseases. BSA is a universal blocking reagent because it does not affect the functions of other
proteins that do need it for stabilization. Because of its negative charge, Bovine Serum Albumin:
Binds water, salts, fatty acids, vitamins and hormones and carries these bound components
between tissues and cells. Bovine serum albumin and gamma globulin are used instead of
human proteins because they are more abundant and give similar results if we were to have

2. used more expensive human proteins. Image 2 shows what the 3 structure of BSA and gamma
globulin look like.
Image 2: The image on the left shows the 3D model of bovine serum albumin. The pink swirls
represent alpha helices in bovine serum albumin (Bujacz, Zielinski, Sekula, 2014).The image on
the right shows the 3D model of part of gamma globulin. The yellow arrows indicate beta
strands (Wang, Ekiert, Ahmad, et al. 2013).
Reducing conditions using betamercaptoethanol breaks up the disulfide linkage in proteins. The
reducing and non-reducing conditions change the three dimensional protein structure. Gamma
globulin will be broken up into two heavy chains and two light chains under reducing condition.
We hypothesize that gamma globulin will be broken up less once amyloid fibril structure is
formed because the disulfide linkages will be buried inside the aggregate. We will able to
compare the accessibility of the disulfide linkages in gamma globulin before and after
aggregation. We hope to apply the knowledge to find agents that break apart the amyloid fibrils
that cause diseases such as Parkinson’s, Alzheimer’s, and type two diabetes.
Research methodology
Bovine serum albumin, is $306.50 (catalog # A7030-50G), will be purchased from Sigma
Aldrich. Gamma globulin, is $198.50 (catalog # G7516-10G) and betamercaptoethanol, is
$27.80 (catalog # M6250-10mL), will also be purchased from Sigma Aldrich. A 10 pack of PAGE
gels will be purchased from BioRad at a cost of $110.00.
We will incubate bovine serum albumin and gamma globulin at pH 2.5 at room
temperature for 24 hours. We will be using SDS PAGE and native PAGE to analyze the proteins
before and after the incubation. However we will mostly be using the SDS PAGE when it comes
to investigating the reducing and non-reducing conditions. For example we will be reducing and
non-reducing conditions using betamercaptoethanol to break up the disulfide linkage in proteins
before and after the aggregate formation.
Expected outcomes of project

3. We hypothesize that we can change the 3D structure of Bovine Serum Albumin and Gamma
Globulin by incubating them at pH 2.5 to form amyloid fibrils. By using reducing conditions
using betamercaptoethanol, we expect gamma globulin to break up into two heavy chains and
two light chains. Bovine serum albumin will not be affected because it is composed of only one
chain and there are no disulfide linkages present. After aggregation of gamma globulin, we
expect to see less of heavy and light chains due to the reduced accessibility of the disulfide
linkages within the protein aggregate.
Schedule and expected completion date:
The project will begin as soon as the funding is obtained. First we will prepare four different
samples using the bovine serum albumin and gamma Globulin, which will be incubated at
different temperatures and combines with different pH buffer. We will be running about 5
different gels in each the Native and SDS solution, or as many as time allows until we are
satisfied with our data. After the gels are complete, the next step is to analyze the gels. This
experiment is expected to be completed by March 2017.
Description of student readiness for proposed project
As an undergraduate student at Winona State University, I have completed laboratory courses
in principles of chemistry, organic chemistry, physical chemistry, physics, as well as biology. I
have worked with many instruments such as the NMR, IR and GC mass spectrum machines.
However I have not used the PAGE gel before this experiment, I have started using the PAGE
gels and analyzing them in Dr. Lee’s research lab in spring of 2016. I wrote a progress report on
using the PAGE gels and what my finding were. I have also read many scholarship articles on
the subject and I feel that as a senior chemistry major, I am well trained on the instruments and
ono the background knowledge.
Statement of where and how results will be presented
This research will be followed up by a poster presentation of the results at Judith Ramalay
Spring Research Symposium at Winona State University, as well as the possible poster
presentation at the ACS National Meeting in March 2017. As well as a poster presentation this
research will presented in a final written report.
Bibliography
1. Tycko, R., 2002. Biochemistry 42:3151-59
2. Bujacz, A., Zielinski, K., and Sekula, B. 2014. Proteins Proteins: Structure, Function, and
Bioinformatics 82, 2199–2208.
3. Wang, F., Ekiert, D. C., Ahmad, I., Yu, W., Zhang, Y., Bazirgan, O., Torkamani, A.,
Raudsepp, T., Mwangi, W., Criscitiello, M. F., Wilson, I. A., Schultz, P. G., and Smider, V. V.
2013. Cell 153, 1379–1393.
4. Ritter, C., Maddelein, M. L., Siemer, A. B., Luhrs, T., Ernst, M., et al. 2005. Nature 43 5:844-
48
5. Nelson, R., Sawaya, M. R., Balbirnie, M., Madsen, A. O., Riekel, C., et al. 2005. Nature 43
5:773-78