Practical 4 sec

HBC 1019 Biochemistry 1 Trimester 1, 2012/2013

Practical 4: Protein Separation; Size Exclusion Chromatography (SEC)

Objectives

1. To learn basic gel filtration chromatography techniques.

2. Compare and contrast the use of different types of column chromatography in

the purification of protein.

3. Explain how naturally occurring or recombinant proteins are separated and

purified using column chromatography.

4. Discuss how the structure and biochemical properties of proteins relate to

purification using column chromatography.

5. Apply the scientific method to solve problems.

Introduction

Chromatography is commonly used in biotechnology for separation or purifying

biological molecules, like proteins. The principle of chromatography that involve with

mobile phase (solvent and the molecules to be separated) and stationary phase either

paper (in paper chromatography), or glass beads, called resin (in column

chromatography), through which the mobile phase travels. Molecules travel through

the stationary phase at different rates because of their chemistry. However, different

medium may have different way to perform the separation technique.

Some of the common types of chromatography are gel filtration chromatography,

affinity chromatography and ion exchange chromatography.

In gel filtration chromatography, commonly referred to as size exclusion

chromatography (SEC), microscopic beads which contain tiny holes are packed into a

column. When a mixture of molecules is dissolved in a liquid and then applied to a

chromatography column that contains porous beads, large molecules pass quickly

around the beads, whereas smaller molecules enter the tiny holes in the beads and

pass through the column more slowly. Depending on the molecules, proteins may be

separated, based on their size alone, and fractions containing the isolated proteins can

be collected.

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The mass of beads within the columns is often referred to as the column bed. The

beads act as “trap” or “sieves” and function to filter small molecules which become

temporarily trapped within the pores. Larger molecules pass around are “excluded”

form, the beads. Each column provided in this practical is prefilled with beads that

effectively separate or “fractionate” molecules that are below 60,000 daltons. As the

liquid flows through the column, molecules below 60,000 daltons enter the beads and

pass through the column more slowly. The smaller the molecule, the slower they

move through the column. Molecules greater than 60,000 pass around the beads and

are excluded from the column – also referred to as the exclusion limit.

This practical is made for the purpose to separate color molecules BSA (molecular

weight of 67,000 daltons, blue in colour) and vitamin B12 (molecular weight of 1,350

daltons, yellow in colour) to illustrate the principle of SEC. The process of separation

of these molecules can be visualized easily as they pass through the chromatography

column.

Materials, reagents and equipments

Protein Mix that contains BSA (Bovine Serum Albumin) and Vitamin B1

Size exclusion chromatography columns

Column buffer

12 Collection tubes

Test Tube rack for holding 12 tubes

Pipette (1ml)

Methods

12 collection tube were placed in the test tube rack. 10 of the collection were

labeled sequentailyfrom 1 – 10. The other two remaing were labled waste and

column buffer .

4 | of culmn buffer wre pipette into the tube labled column buffer

The seal of the both ends of the chromatography column were removed. The

entire buffer wasdraind into the waste collection tube.

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The column was placed gently onto the colletion tube 1. The column should not

jammed tightly into collection tube as the column will not flow. The protein

sample was ready to be loaded onto the column.

The end seal from the column was removed. The top of the column bed was

observed to make sure all of buffer had drained out from the column.one drop of

protein mix was loaded carefully onto the top of the columnbed. The pipette was

been inserted into the column and the drop are loaded just above the top of the

column so that minimally disturb the column bed.

The protein mix was allowed to enter the column bed. 300 ml of column buffer

were added carefully to the top of the column. The drops was started to be

collected into tube 1.

When all the liquid had drained out from the column, another 300 ml of column

buffer were added to the top of the column. The buffer was added as before by

placing pipette just above the top of the column. The protein mix had entered the

column far enough therefore the slight disturbances to the column bed would not

affect the separation. The column was transferred to test 2 and drop was counted.

5 drop of buffer were collected in the test tube 2.

When5drops had been collected into tube 2, the column was transferred onto tube

3. 5 drops of the buffer were collected into each of the collection tube. When 5

drop had been collected into a tube, the column was lifted to the next tube.

For the final tube 10 drops of the buffer was collected. All column were caped

when finished collecting the drops. The sample and column were stored according

to the tutors 'instruction'

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Results and observations

Tube 1 Pink Fast

Tube 2 Pink Fast

Tube 3 Yellow Slow

Tube 4 Yellow Slow

Tube 5 Yellow Slow

Tube 6 Yellow Slow

Tube 7 Yellow Slow

Tube 8 Yellow Slow

Tube 9 Yellow Slow

Tube 10 Yellow Slow

Discussion

The two differnet column chromatography techniquesare gel filtration

chromatography (SEC), affinity chromatography and ion chromatography.

Gel filtiration (SEC) is being used in this lab activity.

Microscopic beads wich contain tiny holes are packed into a column, when a mixture

of molecules is dissolved in a liquid and then applied a chromatography that contain

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porous beads. Large molecules pass quikly around the beads, whereas smaller

molecules enter the tiny pores in the beads and pass through the column more slowly.

Depending on the molecules, proteins might pass quicklyand sparated based on their

size alone fraction containing isolated protiens can be collected.

BSA would be excluded while vitamin B12 is fractionated. BSA will pass more

quickly through the column and appear in the early collectiontubesof fractio. Vitamis

are fractionated by the column. Vitamin will penetrate the pores of the beads

becoming temporarily trapped as a result they pass much slower and appear in the

later fraction.

More buffer are added after the protein mixture was laded onto column in order to

keep the BSA and vitamin mixture in a fluid phas. This ensure that the fluid phase ill

move through the column resulting in completion of the separation process.

BSA molecule exited the column first, and yes this is molecule is much larger of the

two

Based from the situation given, myosin will be the first molecules to appear as it is the

largest followed by hemoglobin as it is the intermediate and the third molecules to

appear is the myoglobin as it is the smallest.

Conclusion

BSA excited the column fisrt followed by vitamin B1.BSA is much larger than

vitamin B1 and so it would be expected to exit first from the size exclusion column.

Reference

www.main.uab.edu

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http://www.main.uab.edu/

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