SDS PAGE is the electrophoresis technique which is used for separating protein

1. BIOINSTRUME
NTATION
ASSIGNMENT
Name: K.V.Thenisha
Class and year:1st year B.Sc biotechnology
Semester:1st semester
Topic: SDS-PAGE

2. 1. Introduction
2. Principle
3. Stock solutions
4. Instrumental setup
5. Procedure
6. Application
7. Limitations
8. Advantage
9. Disadvantage
10. Conclusion
11. Bibliography

3.  SDS-PAGE is the most commonly used gel electrophoretic
system for analyzing proteins.
 This method is based on the separation of proteins according to
size and can also be used to determine the relative molecular
mass of proteins.
 SDS-PAGE: Sodium dodecyl sulphate-Polyacrylamide gel
electrophoresis

4.  Sodium dodecyl sulphate(SDS) is an anionic detergent. When SDS is
added to a solution containing protein, the detergent molecules bind
with the polypeptide of the protein.
 Thus, number of SDS molecule bound to the protein is nearly half of
the number of amino acid residues in the protein.
 The SDS-protein complex has some net negative charge. Therefore,
protein move towards the anode depending on their size.
 Mobility of the protein molecules depends on the net charge, size
and shape of the molecule.

5.  Acrylamide solution in a suitable concentration and in the presence of
ammonium persulphate, solidified into a gel.
 Addition of bisacrylamide into this solution produces cross linking and
sieving gel is formed. Since the separation of molecules depend on
these sieves, the degree of sieving can controlled by regulating the
amount of these chemicals.
 The SDS is a negative charged detergent(Ionic detergent). It is used to
neutralize the effect of charges on proteins and hence only size
becomes the determining factor in the separation.

6.  SDS binds to the hydrophobic regions of protein molecules and
causes them to unfold into extended polypeptide chains.
 SDS cancels the effect of positive charge or protein molecules.
 Mercaptoethanol is added to break the S-S linkage of protein
between subunits.
 SDS-PAGE is used to separate protein and also for molecular weight
determination.

7.  1) Acrylamide (22.2%): 44.4g acrylamide+ 1.2g bis-acrylamide in
200ml distilled water.
 2) Upper gel buffer: Dissolve 6.06g Tris in 80 ml of water. Add
conc.HCL to bring pH to 6.8 and make 100 ml with water.
 3) Lower gel buffer: 18.2g Tris in 80 ml of water,bring pH to 8.8;
make 100 ml of water.
 4) SDS(10%): 1g in 10ml of water and heat at 60 degree Celsius to
dissolve.
 5) Stacking gel: Acrylamide solution- 2.7ml+ water-12ml+ upper gel
buffer- 5 ml+SDS- 0.2 ml + TEMED- 10 ml + Ammonium
persulphate -0.1ml

9.  The vertical electrophoresis unit consists of two buffer tanks, two
electrodes, one slider and a power pack.
 One buffer tank is called upper tank. Both these tanks are made of
glass.
 The lower tank is rectangular and above 30cm in height. On one
side, in this tank, there is a runner for the the upward and
downward movement of the slider. At the bottom, there are two
raised glass mounts to hold the spacer at 1cm height.

10.  The upper tanks are similar in size, but the height is 5cm or 10cm
only. It is located laterally above the level of lower tank. If the slider
is inserted into lower tank, it’s upper portion comes to the bottom of
the upper tank.
 The slider consists of two glass plates and a spacer plate along the
margin between the two plates. This set-up is held together by
bulldog clips. The slider is in the right size to fit into the tanks.
 The space between the glass plates forms a chamber to hold the gel.
There is a comb at the top of the slider to make wells in the gel.
 One electrode is placed horizontally at the bottom of the lower tank.
The other electrode is placed at the bottom of the upper tank.
 These two electrodes are connected with the power pack to provide
enough current supply.

11.  Two dry clean glass plates are taken and a spacer plate is kept
between them along three sides. This setup is held together with
bulldog clips. Petroleum jelly or 2% agar or silicon grease is spread
around the edges of the spacer. This set up is called slider
 The separation gel is prepared and poured into the space between
the two glass plates. Some amount of water is poured on the top of
the gel and gel is left as such for 30 to 60 minutes for
polymerization.
 Stacking gel is prepared. Water is removed from the separating gel
and the gel is once again washed with stacking gel. Then stacking
gel is poured on the separation gel in slider. A few drops butanol is
added to create an even gel slab. Then the gel is dried by blotting
with filter paper.

12.  The comb is inserted in the stacking gel. It is allowed for
solidification for 30-60 minutes.
 The comb is removed carefully without disturbing the well shape.
 The slider is fixed into the lower tank. Necessary quantity of
running buffer is poured into the tank of the electrophoresis
appeeature.
 The protein sample is boiled with SDS buffer for 3 min and cooled to
room temperature. The denatured protein sample is loaded into the
wells at different concentrations. A few wells are loaded with
standard makers. Bromo phenol blue is added to each well as a
marker to locate the molecular movements.

13.  The upper tank is filled with upper gel buffer.
 The electrodes connected to power pack and 80V current are given
for 7-8hrs.
 When the sample reach 1 cm above the lower surface of separating
gel, the power is switched off.
 The slider is removed from the chamber. The gel is carefully
removed and kept into the stainer for 2 hours. Then it is washed
with destainer until the background is clear. Clear blue bands can
be observed in the gel. They are compared to the standard for
confirmation.

15.  SDS- PAGE is used to separate protein based on their mass and size.
 It is useful to determine the molecular weight of protein.
 It is used to separate the different subunits of complex proteins.
 This method can resolve thousands of protein in a biological sample.

16.  The process is difficult as it involves so many steps.
 The preparation of Polyacrylamide gel takes a long time.
 It has toxic monomers.
 Gels should be prepared carefully as they can often leak.
 The gel cannot be used twice in each experiment.

17.  Migration of the protein molecules is proportional to its molecular
weight.
 SDS – PAGE is a highly sensitive test that separate molecules that
have a minimum (~2%) difference in mass.
 Even small amount of sample is enough for processing.
 Pure DNA can be recovered from the gel.
 The pore size of the polyacrylamide gel can be controlled by
adjusting the concentration of the two monomers.

18.  Gels are often difficult to prepare and takes a long time.
 Monomers used are potent neurotoxin chemical.
 Resolution of band is poor due to high alkaline operating pH.
 New gel is needed for each experiment.

19.  SDS- PAGE is a technique that used to separate protein according to
their molecular size through the gel.
 Proteins unfolded and migrate from cathode to anode terminal at
different rates.
 Molecular weight is determined by comparing the result with a
standard curve of relative motility of standard proteins

20.  Help from internet, following website links have been used in the
completion of this assignment
 https://www.iitg.ac.in
 https://microbiologynotes.org
 collegedunia.com
 thesciencenotes.com
 Following book is used to have an idea about the SDS-PAGE:
 Biotechnology, author: V. Kumaresan