BL/CH 401 Lecture 6
Protein/Enzyme Purification -- PART I
Section D

Separation of Proteins by Molecular Size

Imagine the proteins as spheres of different sizes, then size is related to Molecular Weight (MW)

{*Figure 8*}

The larger the sphere, the greater the Molecular Weight or Molecular Size

Thus, proteins can be separated like particles in a sieve -- a molecular sieve which is done with gel beads and often called GEL FILTRATION

Also called size exclusion chromatography .

Illustration of how Gel Filtration works to separate a mixture of different size proteins:

{*Figure 9*}

Column filled with porous, hydrated Gel beads with buffering flowing through it.

First a mixture of two proteins (one small and one large) is applied to column,

Next the small protein pass into buffer inside the Gel beads, while large one passes by Gel bead.

Finally, since the small protein included in the Gel beads is retained by the column,

the large protein elutes first since it was excluded from the inner volume of the Gel beads.

{*Figure 10*}

Details of Gel Bead and Gel Filtration Process:

Gel bead has molecular size holes so that small molecules like water and buffer enter it completely.

Some proteins are small enough to also enter the molecular holes in the Gel bead.

Other proteins are too large to enter holes and pass by the Gel bead.

Concept is of Reverse Sieve since normal sieve retains large and passes small particles.

In Gel Filtration, larger proteins elute first, medium sized ones next and finally smallest proteins elute last.