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Size exclusion chromatography Separation of molecules on the basisof size (and shape)
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Column Parameters
Vs= volume of solvent heldin the pores. This is normallyapproximated to:
Vt-Vo = volume of beads
Vo = void volume
Elution volume of a large“totally excluded” molecule
such as blue dextran
Vt = total volume
Physical volume ofcolumn
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Matrix Types
Sephacryl Protein• Sephacryl™
Cross-linked dextrans
Gel filtration matrices are constructed from a
variety of materials:
S-100 1-100
S-200 5-250
S-300 10-1500
S-400 20-8000
• Sepheroseagarose
• Superdexmixture
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Elution Profile
Ve = Elution volume
(volume of solvent between injection and elution)
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Calculation of Ve
Ve = Vo + Kd (Vs) or
= Vo + Kav (Vt-Vo)
Kav = proportion of
pores available to themolecule.
0
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Running the column• Sample size / Fraction size
▫ 0.5 – 5% of total bed volume (Vt).
▫ Concentration limited by viscosity
• Running time▫ Slow rates allow efficient partitioning into pores and
thus increase resolution
▫ Slow rates increase diffusion of sample on column thus
increasing peak width and reducing resolution.
▫ Protein about 5mL cm-2. h-1
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Running the column
• Buffer▫ Buffer conditions are varied to suit the sample
ype or e requ remen s or ur erpurification since buffer composition does not
directly affect resolution
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Effect of flow rate and bed height onresolution and gel filtration chromatography
Good resolution1
1
Bad resolution2 3+
Effect of bedheight
2
3
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Determination of Molecular Weight
• Calibrate column with known
standards▫ Apply a mixture of proteins on a gel
filtration
106 Da3x105 Da
105 Da104 Da
column
▫ Collect fractions; Do not change
buffer composition
▫ Determine proteins in eluate using
suitable assay
Ve (ml)
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Determination of Molecular Weight
• Calibrate column with known
standards▫ Estimate approximate MW of
unknown protein (Px) using calibration
106 Da3x105 Da
105 Da104 Da
curve with pre-run standard proteins of
known MW by plotting Kav against log
MW
▫ The relationship between the Kav and
log molecular weight is linear over some
range
K a v
Log MW
Ve (ml)
Calibration curve
Px
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Determination of Molecular Weight
Gel Filtration Elution Volumes as a Function of Molecular Weight
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GFC Is useful for desalting proteinsolutions (vs. dialysis)
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Electrophoresis• Separation of proteins, by the differential
migration through a gel according to the size
and charge of the molecules in an electrical
field.
• Examples of gels used are starch, acrylamide,
agarose or mixtures of acrylamide and agarose.
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• Proteins like all other molecules are amphoteric,
they carry positive or negative charge depending
on pH medium and isoelectric point (IP).
Electrophoresis
[H+][OH-] [H+]
pI ~5
Protein becomes increasingly -veProtein becomes
increasingly +ve
[OH-]
At pH < pIthe proteinwill be +ve
At pH > pIthe proteinwill be -ve
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SDS PAGE
•
◦
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SDS PAGE: PROCEDURE
• Proteins to be analyzed are
solubilized and denatured by
boiling (or heating) in the
presence of SDS and reducing
reagent (β-ME or DTT)
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Stackinggel (4%)
Apply Treated protein/dye samplesinto polyacrylamide gel wells
Run the electrophoresis until dyereaches the end of the gel
SDS PAGE: PROCEDURE1 2
Resolvinggel
Remove the gel from theapparatus and stain forproteins
3
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SDS PAGE: Protein Detection
Detection Limit: 50 ng 1ng 10 ngFixing Fixing Non-fixing
Coomassie Blue Silver Sypro Ruby
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• Fraction profiling and determining
sample purity:
SDS PAGE: Application
Screen fractions during protein
purification
Pure proteinPure protein
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• Quaternary structure profile:
SDS PAGE: Application
Comparison of the protein bands
obtained under non reducing and
reducing conditions provides
information about the molecular
size of subunits and protein
complexes
A
B
C
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• MW estimation:SDS PAGE: Application
Separation of molecular weightstandards and sample (unknown
protein).
Calibration curve for molecularweight estimation.1
2
Rf: Migration distance of band
Migration distance of dye front
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MW estimated by SDS-PAGE is
only approximate and referred
• MW estimation:
SDS PAGE: Application
to as .
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SDS PAGE: Application
• SDS gels can be used as a micro-
purification step and the individual
polypeptides can be isolated from
the gel by electroelution or
electroblotting and the amino acid
sequences can be determined or
peptide maps obtained.
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THANK YOU !