bch 530 lecture_student

8/16/2019 BCH 530 Lecture_student

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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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• 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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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:


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:


to as .


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• 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 !

bch 530 lecture_student

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