2012-2013 FALL SEMESTERS

M.U. DEPARTMENT OF BIOENGINEERING

CHEM 213
BIOCHEMSTRY
Experiment 2
SDS Polyacrylamide Gel
Electrophoresis of Proteins
1
GZDE MANO
150810022
DATE OF THE EXPERIMENT

09.10.2012

DATE OF SUBMISSION OF THE REPORT:

16.10.2012

SUBMITTED TO:

Assoc. Prof. Dr Berna SARIYAR

AKBULUT

RESEARCH ASSISTANTS

zlem ATE
Ceyda KULA
Selen DURMAZ

AM

Theory and Introduction

The methods of electrophoresis are used to analyze proteins and nucleic
acids. Matrices are used to separate charged particles in electrophoresis
method. These are several types of matrices. Different type of matrix is
used for DNA or protein. Matrices should be chemical neutral and stable.
The Polyacrylamide matrix is commonly used separating of protein
molecules. It is a kind of polymer matrix. Polyacrylamide is synthesized out
of monomers acrylamide and N, N-methylene-bis acrylamide by a
polymerization reaction. Tetramethylenediamine (TEMED) is induced to
carry out by ammonium persulphate (APS). In addition the polymerization
solution is used in this process. This solution includes acrylamide, N, Nmethylene-bis acrylamide, Tetramethylenediamine (TEMED), ammonium
persulphate (APS). Firstly, APS is added in the solution, APS releases free
radicals so acrylamide molecules are activated. Successive acrylamide
molecules and activated acrylamide molecules react with one another and
long polymer chains occur. Polyacrylamide long chains are cross linked by
bis-acrylamide so network of acrylamide occurs. This network has a
specific pore size. For example; PAGE is not used in DNA separating.
Because, DNA molecules are large size but, pore size of PAGE is small.
Agarose gel electrophoresis is generally used for separating large molecule
of DNA. There are two types of PAGE: Native PAGE and SDS (Denaturing)
PAGE. Native PAGE observes the proteins tertiary and quaternary
structure. The number of subunits, the number of 3-D structure and the
molecular weight of subunits are determined according to this method.
The native PAGE experiment should be followed by the denaturing PAGE
method. SDS (Denaturing) PAGE, the protein subunits are separated by
heating and the help of non-ionic detergent of SDS. The protein subunits
and SDS bind together so protein is negatively charged. Therefore, all
protein subunits can migrate in an electric field. Consequently, molecular
mass of unknown protein can be determined according to known subunit
size or molecular mass (Mr) of proteins. Mobility of unknown protein is
compared to the standard curve by the known protein (Mr). [1]
Preparation of gel: There are many different systems for the
electrophoresis. SDS PAGE has a pre-cast gels for protection the hazard
effect of acrylamide. Preparation of gel needs casting two different layers
of acrylamide between glass plates. Gels are poured between two glass
plates by the help of micropipette. The lower layer that is called separation
gel part is responsible for the separation of proteins by size. The upper
layer that is called stacking gel part is responsible for occurring of wells.
Comb is placed on this upper layer. When the preparation of separating

gel, TEMED is added to the last because, TEMED begins the

polymerization. After polymerization of separating gel, prepared stacking
gel is poured on gel. Finally, comb is placed on the gels. After cooling,
comb is removed out of gels.

Materials and methods

Materials
Gel pouring apparatus (glass plates, the comb and electrophoresis
tank)
Micropipettes and tips
Falcon tubes
SDS PAGE Buffers and Solutions
o Solution A (Acrylamide/Bis; 30:0,8)
Per 50 ml of solution, complete with deionized and distilled
water
Acrylamide 14,6g
NN-bis-methylene-acrylamide 0,4g
Store at dark and 4
o Solution B (4X separating gel buffer, 1,5M Tris-HCl, pH 8,8)
Per 50 ml of solution, complete with deionized and distilled
water
Tris-base 9,25g
Adjust pH to 8,8 with 5N HCl and store at 4
o Solution C (4X stacking gel buffer, 0,5 Tris-HCl, pH 6,8)
Per 25 ml of solution, complete with deionized and distilled
water
Tris-base 1,5g
Adjust pH to 6,8 with 5N HCl and store solution at 4
o %10 APS
Per 5 ml of solution, complete with deionized and distilled
water
Ammonium persulfate 0,5g
o SDS-PAGE running buffer (g/L)
Glycine 15,4g
Tris-base 3,0g
SDS 1,0g
o Electrophoresis separating gel (10 %)
Solution A 4ml
Solution B 2,5ml
10 % APS 50l
TEMED 5l
H2O
d
3,5ml
o Electrophoresis stacking gel (5 %)

Solution A 0,67ml
Solution C 1ml
10 % APS 50l
TEMED 5l
H2O
d
2,5ml
Methods
1. Glass plates that were used to pour gels were cleaned with distilled
water.
2. Glass plates were cleaned with 70 % Ethanol.
3. Lastly, glass plates were cleaned with isopropanol and they were
dried.
4. Glass plates were piece together and they placed in the gel pouring
apparatus. Open section between the glass plates must be inside of
the gel pouring apparatus.
5. Firstly, separating gel was prepared. 3,5ml distilled, 4ml solution A
and 2,5ml solution B and 50l 10% APS were added in the falcon
tube by the help of micropipette. TEMED was added in the falcon
tube lastly. Because, TEMED begins the polymerization.
6. Separating gel was poured in glass plates by the help of
micropipette. 500 l isopropanol was added this gel. Because, gel
must polymerize smoothly. Polymerization of the gel was waited.
7. Secondly, stacking gel was prepared. 2,5ml distilled water, 0,67ml
solution A, 1ml solution C and 50l APS were added in the falcon
tube. Before adding of stacking gel on the separating gel,
isopropanol was removed on the separating gel. After removing of
isopropanol, stacking gel was added on the separating gel by the
help of micropipette.
8. The comb was placed between glass plates that consist of
separating and stacking gels.
9. When the polymerization occurs, the comb was removed on the gel,
wells occur.
10.
Gel was waited in distilled water so that it is not dry up to next
week.

Results
Gel was waited for the polymerization. After the polymerization, the
comb was placed in gel.
After occurring wells, SDS-PAGE gel was waited up to next week.
Because, gel will be used to next week for loading of proteins in the
wells.

Discussion

 When the separating gel was added between the glass plates,
polymerization was waited for a while. However, gel did not
polymerize. This process took a long time. It did not occur in
expected time. The reason for this, APS amount may be less.
Because, APS is catalysts for this process. TEMED amount may be
less. Because, TEMED is necessary for the polymerization. It begins
the polymerization. For all this reasons, gel cannot polymerize.

References
[1] Dr. A. J. Nair, Principles of Biotechnology, first edition 2007, LAXMI
PUBLICATIONS (P) (LTD), page 256-257-258
[2] Introduction to SDS-PAGE, David R. Caprette, Rice University Dates
http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2.html