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Laboratory Activity Seven

Proteins – Gel Filtration Chromatography

& Estimation of Molecular Weight

Introduction to

Gel Filtration Chromatography(Also known as “size exclusion chromatography”)

From lab five . . . All chromatography techniques have three components:

1. Stationary phase2. Mobile phase3. Solute/sample phase4. Column (container)

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Special Characteristics of Gel Filtration Chromatography

A form of “partition chromatography”. Stationary phase is a porous gelatinous

matrix (in the form of beads). Sample components enter pores and are

temporarily (reversibly) trapped/retarded as mobile phase moves.

Relative mobility through column is dependent on size of pores vs. size of solute molecules.

Larger molecules exit column faster than smaller molecules – i.e. separation is based on molecular weights.

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From before . . . Other Chromatographic Separation Techniques

Gel Filtration(aka size exclusion)

Ion Exchange Affinity

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Hypothetical Elution Profileof a Crude Protein Extract

Gel filtration chromatography can be used to purify proteins.

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Purpose(s) of Lab 07 Introduction to the theory and practice of gel filtration chromatography. Calibration & use of a sephadex G-75 column for the separation &

recovery of two known proteins. Create a simple (2-point) Kav vs. log10MW graph to estimate the MW of a

hypothetical protein.

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Dextran vs. Sephadex®

H2C CH CH2Cl

O

Epichlorohydrin

+

Dextran(-1,6-glucose w/ -1,3 branches)

Sephadex®

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Calibration of a Sephadex Column

Defining Features of a Gel Filtration Colum: Total column volume (Vt) Interstitial volume (Vi) Void volume (Vo)

These parameters must be determined before a column can be properly used.

Process is referred to as “calibration”.

Vt Vi Vo

Vt & Vo are experimentally determined; Vi is calculated:

Vi = Vt – Vo

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Determination of Vo & Vt

Vo: Apply a very high MW substance (blue dextran*). Substance is completely excluded from pores. Determine the volume of mobile phase required

to elute the substance from the column.

Vt: Apply a very low MW substance (DNP-glycine*). All molecules enter pores, results in maximum

retention. Determine the volume of mobile phase required

to elute the substance from the column.

*MW of blue dextran = 2,000,000.*MW of DNP-glycine = 240.

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Sample Calibration of a Sephadex Column

Elution Volume / Fraction No.

OD

400 (

——

)

OD

620 (

– –

–)

(Blue Dextran)

(DNP-amino acid)

0 5 10 15 20 3025 35

(DiNitroPhenyl-Glycine)

(Cibacron Blue 3G-A)

Vo Vt

Vo = 10 mL, Vt = 29.5 mLVi = Vt – Vo

Vi = 29.5 – 10 = 19.5 mL

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Why Calibrate a Column ? The MW of a given protein molecule is related to its elution volume (Ve);

(high MW proteins elute with less volume than low MW proteins). Elution volumes can be used to predict the MW of unknown proteins.

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Molecular Weight Estimation

Procedure:1. Calibrate column to determine Vo & Vt.

2. Determine Ve’s of a series of known proteins/MW’s.

3. Determine the Ve of the unknown protein.

4. Calculate the Kav of all proteins.

5. Make a graph of Kav vs. Log10[MW] of all proteins.

Kav =Ve – Vo

Vt – Vo

Kav “partition coefficient”It represents the fraction of the stationary phase that is available for diffusion of a given solute (i.e. protein).

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Hypothetical Plot of Kav vs. Log10[MW]

4 5 6

0

0.5

1.0

Log M.W.

K av

= standard proteins= unknown protein

If log10MW of unknown protein = 5;Then MW of unknown protein = 105 = 100,000.

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Practical Considerations There are many different kinds/brands

of gel media (see appendix XV): Variations in chemical composition. Variations in MW ranges & exclusion limits

Sephadex is “biodegradable”; must be kept sterile.

Gravity flow is inexpensive; pressurized flow systems are expensive.

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Lab 07 Chromatography System

Simple gravity flow. Fraction collector set at 25 drops 1 mL.

Before getting started: Turn on spectrophotometer; program for

dual absorbance (400 & 620 nm; see appendix I.D).

Make 1 mL of a 200-fold(+) dilution of blue dextran/DNP-AA calibration mix. (10 & 5 mg/mL 50 & 25 µg/mL)

Measure ABS @ 620 & 400 nm. ABS/mL will facilitate recovery estimates.

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USE CautionWhen Loading & Running Columns

Procedure: Allow excess elution buffer to enter column with meniscus at bed surface. STOP Very carefully apply sample onto bed surface (along column edge). Allow sample to enter column (as above). STOP Apply fresh elution buffer onto bed surface (as above). Allow elution buffer to enter column AND start collecting fractions. Fill/replenish reservoir as needed. Collect fractions until eluant is colorless; continue rinsing w/2 column volumes.

DON’T . . . • Disturb the bed surface.• Let columns run dry.

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Separation of BSA/Lysozyme Mix MW BSA = 67 kD; MW Lysozyme = 14.4 kD. Concentrations = 4 mg/mL each. Apply 300 µL (as before). Measure ABS @ 280 nm (use UV-220 cuvettes). Determine Ve’s; calculate Kav’s. Predict MW of hypothetical unknown. Estimate recovery of protein (mg/mL = A280 x 1.55).

Questions or Comments

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