Electrophoresis is a technique used to separate and sometimes purify macromolecules - especially proteins and nucleic acids - that differ in size, charge or conformation.

1. Gel electrophoresis Native,
Denaturing & Reducing
By- Lovnish Thakur
ASU2014010100099
Integrated Biotech- 3rd Sem
BSBT-504(Bioanalytical Technique & Instrumentation-II)
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2. Gel electrophoresis
Electrophoresis is a technique used to separate
and sometimes purify macromolecules -
especially proteins and nucleic acids - that
differ in size, charge or conformation.

3. PRINCIPLE
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4. SDS-PAGE(Denaturing gel
electrophoresis)
 . Denaturing the proteins nullifies structural effects
on mobility, allowing separation on a true
charge/mass ratio basis.
 The most commonly used denaturant is sodium
dodecyl sulfate (SDS).
 Proteins solubilized in SDS bind the detergent
uniformly along their length to a level of 1.4 g
SDS/g protein.
 This creates a charge/mass ratio which is
consistent between proteins.
 For this reason, separation on a polyacrylamide
gel in the presence of SDS occurs by mass alone..

5. Material Required
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6. Chemical Requirement
Acrylamide solutions
Ammonium persulfate is used as a catalyst for the
copolymerization of acrylamide and bisacrylamide gels.
SDS (0.1%)
Protein standard molecular-weight markers
Protein samples to be resolved
SDS stock solution for resolving and stacking gels
1× SDS gel-loading buffer
0.2% (w/v) bromophenol blue
20% (v/v) glycerol 200 mM β-mercaptoethanol
TEMED (electrophoresis grade)
Tris-glycine buffers for SDS-polyacrylamide gels.

7. Procedure
Gel casting
• Assemble the glass plates according to the manufacturer’s
instructions.
• Make gel using desired concentration of acrylamide and
bisacrylamide and Ammonium persulfate as a catalyst for
copolymerization.
• Pour the acrylamide solution into the gap between the glass plates.
• After polymerization of resolving gel is complete (30 minutes), pour
off the overlay and wash the top of the gel several times with de-
ionized H2O to remove any unpolymerized acrylamide.
• Pour the stacking gel solution directly onto the surface of the
polymerized resolving gel.
• Immediately insert a clean Teflon comb into the stacking gel
solution.

8. Loading
• After polymerization is complete (30 minutes),
remove the Teflon comb carefully.
• Mount the gel in the electrophoresis
apparatus. Add Tris-glycine electrophoresis
buffer to the top and bottom reservoirs.
• Load up to 15 μl of each of the samples in a
predetermined order into the bottom of the
wells.

9. Run gel
Attach the electrophoresis apparatus to an electric
power supply
• Apply a voltage
• Run the gel until the bromophenol blue reaches
the bottom of the resolving gel.
• Turn off the power supply.
• Remove the glass plates from the electrophoresis
apparatus and place them on a paper towel.
• At this stage, the gel can be fixed, stained with
Coomassie Brilliant Blue or silver salts.

10. RESULT
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11. Reducing
• In Reducing gel electrophoresis
mercaptoetanol is used which assist SDS in
denaturing by reducing the disulphide bond in
protein that is the reason it is called reducing
electrophoresis.
• The formation of nonlinear species can effect
mobility - ie, if you have an intra chain disulfide
bond in a monomer, it may run differently than
the reduced monomer.

12. Native PAGE
• Sometime, we need to separate protein in non-
denaturing conditions. This type of polyacrylamide gel
electrophoresis is also called native gel electrophoresis
because protein remains in native form even after
electrophoresis
Difference between Native & SDS PAGE
• The basic difference in the native gel electrophoresis
(native-PAGE) is the electrophoresis buffer does not
contain SDS.
• Also, loading buffer does not have SDS and reducing
agents and samples are not boiled.
• Rest of the things are similar to SDS- PAGE gel
electrophoresis

13. ADVANTAGE
• Biological activity of protein remain intact
• Native PAGE is used for separation of
enzymes/isozymes.

14. APPLICATIONS

15. Quantification of muscle mitochondria oxidative
phosphorylation enzyme via histochemical staining of blue
native polyacrylamide gel
Summary-
In this blue-Native Polyacryamide gel electrophoresis is used to
separate intact multi-subunit complex of enzyme. For particular
enzyme separation require denaturing PAGE.
In this they demonstrate that they can study enzyme activity using
these technique. And compare the number of mitochondria in
normal and heart cell by quantification of phosphorylation
enzyme.

16. Separation and identification of hen egg protein isoforms using
SDS–PAGE and 2D gel electrophoresis with
MALDI-TOF mass spectrometry
Summary-
High-resolution techniques for proteome analysis, including SDS–PAGE
and 2-dimensional (2D) gel electrophoresis, combined with mass-
spectrometry, were employed to separate and identify several protein
components in hens egg.
An advanced and sensitive glycoprotein staining kit was used to detect
the presence of glycosylated proteins in the egg samples. Numerous
spots were revealed when a mixture of egg white and yolk was
subjected to 2D gel electrophoresis.
Several of the already known egg proteins were identified.
Result- Isoforms of ovalbumin and conalbumin were visualised.

17. Reference
• http://www.ispybio.com/search/protocols/sds%2
0protocol29.pdf
• http://www.protocol-online.org/biology-
forums/posts/12890.html
• https://classes.soe.ucsc.edu/bme220l/Spring11/
Reading/SDS-PAGE-theory.pdf
• http://www.researchgate.net/publication/22338
5062_Separation_and_identification_of_hen_egg
_protein_isoforms_using_SDS-PAGE_and_2-
D_gel_electrophoresis_with_MALDI-
TOF_mass_spectrometry._Food_Chem

18. THANK YOU