Biochem Lab 2: SEC Chromatography

What is chromatography for?

to purify biological components and separate the components in biotech

What is size exclusion chromatography?

aka gel filtration chromatography

uses beads with tiny holes in a column

large molecules pass around beads and smaller molecules get stuck in the beads passing through the column more slowly

proteins are separated by size

fractions containing isolated proteins are collected

what is affinity chromatography?

an antibody (that can bind to the protein) is purified and attached to the beads in the column

proteins of interest will bind to the antibody when going through the column

then another buffer (acid or salt) is used which disrupts the bond and the protein can be collected

What is ion exchange chromatography?

beads have a charge that is opposite of the protein of interest (resin used)

proteins are attached to beads

anion exchange if protein is negatively charged

cation exchange if protein is positively charged

high salt buffer used to get desired protein out of column

What is molecules separated by is SEC?

size

What is the mobile phase in the chromatography column?

mixture of molecules dissolved in liquid which is applied to the column

What is the stationary phase of the chromatography column?

solid support in column of the small microscopic beads

What is the trapa or sieves?

the beads as they act as filter for the smaller molecules that are temporarily trapped in the pores

What molecules are excluded from the beads in the column?

the larger beads

What is fractionated in column chromatography?

8 columns come in a kit that are prefilled with beads

these beads fractionate or separate the molecules that are below 60,000 daltons

What happens to molecules more than 60000 daltons in the column?

pass around and are excluded from the column (exclusion limit)

What happens to molecules less than 60000 daltons in the column?

get stuck in the beads and go through the column slowly

What is buffer used for in SEC?

used to dissolve the proteins/biomolecules to make the mobile phase

What is in the sample in chromatography?

mixture of buffer and the biomolecules

What are the two biomolecules used in our SEC lab?

Hemoglobin and vitamin B12

What color and size is hemoglobin?

brown with a molecular weight of 65,000 daltons (passes around beads)

What is hemoglobins purpose?

main component in red blood cells to transport oxygen to tissues

gives red blood cells their red color

can also bind to carbon monoxide (actually has a higher affinity for it than O2)

When disease is caused by mutation in the amino acid that changes the 3D structure of hemoglobin polypeptides?

sickle cell anemia, causes fatigue or death because of lack of O2

What color and size is vitamin B12?

pink with a molecular weight of 1350 daltons (gets stuck in beads)

What is vitamin B12’s purpose?

essential cofactor in many biochem reactions inside the body like breaking down fats

(in eggs, dairy, and meats, not vegetables/ plants

How does B12 get into your blood stream?

vitamin B12 consumed binds to carrier proteins in intestinal tracts and passes through the intestine into the blood stream

(can be mutations in carrier proteins making people deficient in vitamin B12)

What type of hemoglobin is used in our SEC experiment?

bovine hemoglobin

What is first added to the column in the experiment?

4 mL of column buffer added to the column buffer tube for clean uncontaminated buffer

What is added after the column is placed onto tube 1?

1 protein mix drop onto the column bed

What do you add after protein mix has been added to the column?

250 microliters of column buffer twice to act as the mobile phase to push the protein mixture down through the column bed

What happens after drops are collected into tube 1?

drain all from column and add 3 mL of column buffer and collect 5 drops into each tube (10 on last tube/tube 10)