SDS-PolyAcrylamide

Gel Electrophoresis (SDS-PAGE) Of Proteins

Practical Manual

MDSC 2001: Respiration
Department of Preclinical Sciences
Biochemistry Unit
(Academic Year 2013/2014)

SDS-PolyAcrylamide Gel Electrophoresis (SDS-PAGE)

Of Proteins

SDS-PolyAcrylamide Gel Electrophoresis (SDS-PAGE) is a technique that is used to provide information
on the sub-unit structure and molecular weight of proteins. Although it is an electrical method, it is quite
different to isoelectric focusing as it does not depend on the intrinsic electrical properties of the protein.
Interestingly, this technique reveals similar information to Exclusion Chromatography, which is a non-
electrical method.
One of the early difficulties persons using SDS-PAGE faced was that when the absolute migration was
used different researchers obtained different molecular weights for the same protein. To overcome this
problem it was agreed that the relative mobility of proteins rather than their absolute mobility would be
employed when analyzing SDS-PAGE data. Recall:

In this experiment you will be using SDS-PAGE to analyse a number of proteins. The proteins to be
studied will include two or more of the following. The oxygen binding proteins of hemoglobin and
myoglobin, the electron transport chain protein cytochrome c serum albumin and the well known
Carbonic anhyrase. Hemoglobin has 4 chains, two identical alpha chains 15,126 d and two identical beta
chains 15,867 d. Myoglobin has one chain, 17,000 d and cytochrome c, one chain 12,300 d. You will
notice that these heme proteins unlike non heme proteins, possess characteristic absorbance spectra.
By combining spectral data and the results of SDS-PAGE you will be required to establish the identity
of some proteins.

Method
Part A: SDS=PAGE
You are provided with:
i.

Five Protein Solutions A, B,C,D and E from which you will be assigned TWO.

ii. Denaturing Solution, F.

iii. Molecular weight standards, S.

Denaturing Procedure:
Label two micro tubes A/B/C/D/E appropriately. Into each tube place 100l of the corresponding
solution and add 100l of Denaturing solution. Into a third micro tube add 20l of the standard, S and
add 20l of Denaturing solution. Stack the tubes in a rack and place them in a hot water bath for 10
minutes. The water bath should be maintained at approximately 90oC.
Application:
Using a Hamilton syringe apply 25l of each of the denatured solutions into separate wells of the SDS-
GEL provided, see Fig 1. Record the lane numbers and sample (A/B/C/D/E/S) applied to each lane.
The gel will be run for approximately 1 hour as in Fig 2 after which it is removed from the glass plates
and stained (5 minutes) with a blue dye which binds to the proteins. Finally the gel is destained. In the
destaining process the blue dye is washed out of the gel but the protein bands retain the blue colour.
Additional Information
Composition of Staining Solution: Coomassie Blue R-250 0.2% in 50% methanol, 10% Acetic acid
(Filtered before use)
Destaining Solution: 30% methanol, 10% Acetic acid

Protein

Molecular Weight

Myosin

211,475

-Galactosidase

118,579

BSA

78,995

Ovalbumin

53,045

Carbonic Anhydrase

36,881

SoybeanTrypsin inhibitor

28,643

Lysozyme

17,809

Aprotinin

6,435

Table 1: Protein Molecular weights (Daltons)of Commercial Standard(BIO-RAD Lab Inc., CA,USA)

Figure 1: Proteins being loaded onto gel

Figure 2 :Separation of proteins on fully
assembled apparatus

Part B: Absorbance Spectra (To be done while the gel is running)

Mark two test tubes A/B/C/D/E according to the proteins assigned in Part A. Into each tube place 300l
of one of the solution used in Part A and add 2ml of the buffer provided. Place 2 ml of buffer in a
cuvette, zero the spectrophotometer and record a base line over the wavelength range 400nm to
600nm. Replace the buffer with one of the protein (A,B,C,D or E) solutions and record its spectra over
the same wavelength range. Using the tip of a spatula, add a small quantity of sodium dithionite to the
cuvette and record the spectrum again. Repeat the exercise for the other protein solution. Retain a copy
of your spectra.