Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis (SDS-PAGE)

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Sodium dodecyl sulfate-Polyacrylamide gel
electrophoresis (SDS-PAGE)

• Irene Goh
• Rosarine Metusela

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Objectives

• To use the SDS PAGE analytical procedure to
  identify and/or isolate the following proteins
• Ovalbumin
• Casein
• Gluten
• To be able to understand the principles of gel
  electrophoresis
• To apply and follow safety procedures while
  carrying out the experiment

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What is SDS-PAGE?

• Based on the migration of charged molecules in an
  electric field
• Separation technique
• Uses the Polyacrylamide gel as a support
  matrix. The matrix inhibits convective mixing
  caused by heating and provides a record of the
  electrophoretic run.
• Polyacrylamide is a porous gel which acts as a
  sieve and separates the molecules

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Role of SDS

• Denatures proteins by wrapping around the
  polypeptide backbone.
• SDS binds to most proteins in amount roughly
  proportional to molecular weight of the
  protein-about one molecule of SDS for every two
  amino acids (1.4 g SDS per gram of protein)
  (Lehninger Principles of Biochemistry).
• In doing so, SDS creates a large negative charge
  to the polypeptide in proportion to its length

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Role of SDS (cont)

• SDS also disrupts any hydrogen bonds, blocks many
  hydrophobic interactions and partially unfolds
  the protein molecules minimizing differences
  based on the secondary or tertiary structure
• Therefore, migration is determined not by the
  electrical charge of the polypeptide, but by
  molecular weight.
• The rate at which they move is inversely
  proportional to the molecular mass
• This movement is then used to determined the
  molecular weight of the protein present in the
  sample.

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Procedure materials

• 1.A Mighty Small II, SE 260 Mini-Vertical Gel
  Electrophoresis Unit
• 2.0.5 TrisCl, pH 6.8 solution
• 3.10 SDS solution
• 4.Sample treatment buffer
• 5.SDS glycine running buffer
• 6.β-Mercaptoethanol solution
• 7.Brilliant Blue R concentrate
• 8.Destaining solution
• 9.Precast polyacrylamide separating gel
• 10.Fine tipped microsyringe
• 11.Protein samples (ovalbumin, casein, and
  gluten)

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Procedure solutions

• 0.5M TrisCl, pH 6.8 (4X Resolving gel buffer)
• 10 SDS solution
• 2X Sample treatment buffer
• SDS glycine running buffer
• Destaining solution

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Procedure electrophoresis unit

• Initial preparation-wash the unit
• Preparing the gel sandwich(es)
• ensure that the plates are completely polymerized
  before loading
• Install the gel sandwhich(es) into the unit
  before loading any of the protein samples.
• Loading the protein samples
• Dry sample add equal volumes of treatment buffer
  solution, and deionised water to achieve the
  required concentration. Heat in a tube, in
  boiling water for 90 seconds

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Procedure electrophoresis unit

• Fill upper buffer chamber with running buffer
• Using a fine-tipped microsyringe, load the
  treated protein samples into the wells so that
  the volume in each well is raised by 1mm
• Fill the lower buffer chamber

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Procedure running the gel

• Place the safety lid on before plugging in the
  leads of the unit to the power supply.
• Run the gel at 20mA per gel, using a constant
  current
• When it reaches the bottom of the gel, the run is
  complete
• Turn off the power supply, and disconnect the
  leads, before removing the safety lid

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Procedure running the gel

• Carefully remove the gel(s) from the plates
• Lay it into a tray of staining solution for about
  10 minutes.
• Remove the gel carefully and place it in between
  two layers of transparencies, cut along the edges
  of the gel and analyse the results.

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Results and discussion

• The results discussed here is, the sample results
  which was provided by the supervisor

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Results and discussion
Protein Standard Theoretical MW log10 MW Distance migrated (cm) Relative distance
Aprotinin, bovine lung Aprotinin, bovine lung 6,500 3.812913357 1.65 0.113793103
a-lactalbumin, bovine milk a-lactalbumin, bovine milk 14,200 4.152288344 3.55 0.244827586
Trypsin inhibitor 20,100 4.303196057 4.05 0.279310345
Tyrpsinogen, bovine pancrease Tyrpsinogen, bovine pancrease 24,000 4.380211242 4.55 0.313793103
Carbonic anhydrase 29,000 4.462397998 4.90 0.337931034
Glyceraldehyde-3-phosphatedehydrogenase Glyceraldehyde-3-phosphatedehydrogenase Glyceraldehyde-3-phosphatedehydrogenase 36,000 4.556302501 5.85 0.403448276
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Results and discussion
Protein Standard Theoretical MW log10 MW Distance migrated (cm) Relative distance
Glutamic dehydrogenase, bovine liver Glutamic dehydrogenase, bovine liver 55,000 4.740362689 6.60 0.455172414
Albumin, bovine serum Albumin, bovine serum 66,000 4.819543936 7.65 0.527586207
Fructose-6- phosphate kinase Fructose-6- phosphate kinase 84,000 4.924279286 8.35 0.575862069
Phosphorylase b, rabbit muscle Phosphorylase b, rabbit muscle 97,000 4.986771734 8.75 0.603448276
B-galactosidase, E.coli B-galactosidase, E.coli 116,000 5.064457989 9.75 0.672413793
Myosin, rabbit muscle Myosin, rabbit muscle 205,000 5.311753861 12.40 0.855172414







Glutamic dehydrogenase, bovine liver Glutamic dehydrogenase, bovine liver 55,000 4.740362689 6.60 0.455172414
Albumin, bovine serum Albumin, bovine serum 66,000 4.819543936 7.65 0.527586207
Fructose-6- phosphate kinase Fructose-6- phosphate kinase 84,000 4.924279286 8.35 0.575862069
Phosphorylase b, rabbit muscle Phosphorylase b, rabbit muscle 97,000 4.986771734 8.75 0.603448276
B-galactosidase, E.coli B-galactosidase, E.coli 116,000 5.064457989 9.75 0.672413793
Myosin, rabbit muscle Myosin, rabbit muscle 205,000 5.311753861 12.40 0.855172414
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Results and discussion
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Results and discussion

• the relationship between the logarithm of the
  standards and the relative distance travelled by
  each protein through the gel is linear
• The equation of the line was obtained and used to
  calculate the relative molecular weights (Mr) of
  the samples in lanes b-l of the gel
• x (y 1.7679)/0.4785
• x Mr
• y Relative distance travelled by the sample in
  centimetres

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Results and discussion
Sample lane distance(cm) relative distance log10 Mr Mr (Da)
b (i) 2.5 0.172413793 4.054992253 11349.9057
(ii) 5.05 0.348275862 4.422520088 26455.75061
(iii) 7.9 0.544827586 4.833286492 68121.85908
c 3.1 0.213793103 4.141469391 13850.62563
d 9.15 0.631034483 5.013447195 103144.766
e 5.65 0.389655172 4.508997226 32284.73497
f 4.05 0.279310345 4.278391525 18984.16611
g 8.95 0.617241379 4.984621482 96520.92657
h 11.4 0.786206897 5.337736461 217638.8693
I 4.25 0.293103448 4.307217238 20286.97237
j 3.7 0.255172414 4.227946528 16902.32812
k 7.65 0.527586207 4.797254351 62698.09577
l 4.75 0.327586207 4.379281519 23948.67659

Mr gt Relative molecular weight of the unknown samples. Mr gt Relative molecular weight of the unknown samples. Mr gt Relative molecular weight of the unknown samples. Mr gt Relative molecular weight of the unknown samples.
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Results and discussion

• From the molecular weights obtained for the
  proteins to be analysed in the experiment
• Cassein 24,000 Da
• Ovalbumin 46,000 Da
• Gluten 20,000 11,000,000 Da
• It would be expected that the relative molecular
  weights of these proteins, would be close their
  respective theoretical values shown above.

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Conclusion

• SDS PAGE is a useful method for separating and
  characterising proteins, where a researcher can
  quickly check the purity of a particular protein
  or work out the different number of proteins in a
  mixture.
• Since we did not obtain results for the
  experiment,
• we have to rely on sample results
• Cannot validate the experimental technique