Transcript Sodium dodecyl sulfate-Polyacrylamide gel electrophoresis

Sodium dodecyl sulfatePolyacrylamide gel
electrophoresis (SDS-PAGE)
Irene Goh
Rosarine Metusela
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
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
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 proteinabout 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
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.
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)
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
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
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
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
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.
Results and discussion
 The results discussed here is, the sample
results which was provided by the
supervisor
Results and discussion
Protein
Standard
Distance
migrate
d (cm)
Theoretical
MW
log10 MW
Aprotinin, bovine lung
6,500
3.812913357
1.65
0.113793103
a-lactalbumin, bovine milk
14,200
4.152288344
3.55
0.244827586
20,100
4.303196057
4.05
0.279310345
24,000
4.380211242
4.55
0.313793103
29,000
4.462397998
4.90
0.337931034
36,000
4.556302501
5.85
0.403448276
Trypsin inhibitor
Tyrpsinogen, bovine
pancrease
Carbonic
anhydrase
Glyceraldehyde-3phosphatedehydrogenase
Relative
distance
Results and discussion
Protein
Standard
Glutamic dehydrogenase,
bovine liver
Albumin, bovine serum
Fructose-6- phosphate kinase
Phosphorylase b, rabbit
muscle
B-galactosidase, E.coli
Myosin, rabbit muscle
Theoreti
cal
MW
log10
MW
Distance
migrated
(cm)
Relative
distance
55,000
4.740362
689
6.60
0.455172414
66,000
4.819543
936
7.65
0.527586207
84,000
4.924279
286
8.35
0.575862069
97,000
4.986771
734
8.75
0.603448276
116,000
5.064457
989
9.75
0.672413793
205,000
5.3117538
61
12.40
0.855172414
Results and discussion
Standard curves for proteins with known
molecular weights
0.9
y = 0.4785x - 1.7679
Relative Migration (cm)
0.8
2
R 0.9672 =
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
1
2
3
log10 MW
4
5
6
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
Results and discussion
Sample lane
b (i)
relative
distance
distance(cm)
log10 Mr
Mr (Da)
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 => Relative molecular weight of the unknown samples.
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.
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