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3.  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. 3

4. 4 Gel Filtration (aka size exclusion) Ion Exchange Affinity

5. 5 Gel filtration chromatography can be used to purify proteins.

6.  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) K av vs. log 10 MW graph to estimate the MW of a hypothetical protein. 6

7. 7 H 2 C CH CH 2 Cl O Epichlorohydrin + Dextran (  -1,6-glucose w/  -1,3 branches) Sephadex®

8. 8 VtVtVtVt ViViViVi VoVoVoVo V t & V o are experimentally determined; V i is calculated: V i = V t – V o

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10. 10 Elution Volume / Fraction No. OD 400 (——) OD 620 (– – –) (Blue Dextran) (DNP-amino acid) 05101520302535 VoVoVoVo VtVtVtVt V o = 10 mL, V t = 29.5 mL V i = V t – V o V i = 29.5 – 10 = 19.5 mL

11.  The MW of a given protein molecule is related to its elution volume (V e ); (high MW proteins elute with less volume than low MW proteins).  Elution volumes can be used to predict the MW of unknown proteins. 11

12. 12 K av = V e – V o V t – V o K av  “partition coefficient” It represents the fraction of the stationary phase that is available for diffusion of a given solute (i.e. protein).

13. 13 If log 10 MW of unknown protein = 5; Then MW of unknown protein = 10 5 = 100,000.

14.  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. 14

15. 15  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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18.  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 V e ’s; calculate K av ’s.  Predict MW of hypothetical unknown.  Estimate recovery of protein (mg/mL = A 280 x 1.55). 18

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