Biochem Sds Page

Introduction

SDS-PAGE

SDS-PAGE is an abbreviation for sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. SDS is an anionic detergent, meaning that when dissolved, its molecules have a net negative charge within a wide pH range. A polypeptide chain binds amounts of SDS in proportion to its relative molecuar mass. The negative charges on SDS destroy or denatures most of the complex structure of proteins, and are strongly attracted toward an anode (positively-charged electrode) in an electric field. Polyacrylamide gels restrain larger molecules from migrating as fast as smaller molecules. Because the charge-to-mass ratio is nearly the same among SDS-denatured polypeptides, the final separation of proteins is dependent almost entirely on the differences in relative molecular mass of polypeptides. In a gel of uniform density the relative migration distance of a protein is negatively proportional to the log of its mass. If proteins of known mass are run simultaneously with the unknowns, the relationship between distance travelled and mass can be plotted, and the masses of unknown proteins estimated.

Protein separation by SDS-PAGE can be used to estimate relative molecular mass, to determine the relative abundance of major proteins in a sample, and to determine the distribution of proteins among fractions. The purity of protein samples can be assessed and the progress of a fractionation or purification procedure can be followed. Different staining methods can be used to detect rare proteins and to learn something about their biochemical properties.

The lower layer of the polyacrylamide gels (separating, or resolving, gel) is responsible for separating polypeptides by size. The upper layer (stacking gel) includes the sample wells. It is designed to sweep up proteins in a sample between two moving boundaries so that they are compressed (stacked) into micrometer thin layers when they reach the separating gel

Ion-Exchange Chromatography

Ion exchange chromatography separates compounds based on net surface charge. Molecules are classified as either anions (negative charge) or cations (positive charge). Some molecules may have both an anionic and cationic group. A positively-charged will bind a compound with an overall negative charge. Whereas, a negatively-charged will bind a compound with an overall positive charge. If the protein has more positive charges than negative charges, it is said to be a basic protein. If the negative charges are greater than the positive charges, the protein is acidic. The use of ion-exchange chromatography, then, allows molecules to be separated based upon their charge

To ensure that a protein has a particular net charge, dissolve it in a buffer that is either above or below its isoelectric point (pI). For example, a protein with a pI of 5 will have a net negative charge if it is in a buffer at pH 7. In this case, the protein could bind to a positively charged solid support like diethylaminoethanol (DEAE). Whereas, a protein with a pI of 7 will have a positive charge in a buffer at pH 5 and can bind to a negatively charged anion.

Ion exchange matrices can be further categorized as either strong or weak. Strong ion exchange matrices are charged (ionized) across a wide range of pH levels. Weak ion exchange matrices are ionized within a narrower pH range. The four most common ion exchange chemistries are shown here: Species with a larger net charge will bind to the resin more tightly than species with lower net charge. As net charge is pH dependant, control of pH is crucial.

Objective

Results

Part 1: SDS-PAGE

Table 1: Molecular weights and distance migrated by marker fragments

Band

Molecular Weight (kDa)

log10 Molecular Weight

Distance migrated (cm)

1 260.0 2.415 0.2

2 160.0 2.204 2.6

3 110.0 2.041 4

4 80.0 1.903 5.6

5 60.0 1.778 8.1

6 50.0 1.699 12.5

Figure 1: Graph of distance migrated versus log10 molecular weight

Calculations:

From the graph, the equation Distance Migrated =-15.21(log10 molecular weight) + 36.03.

Sample A:

Distance migrated = 6.6 cm,

Distance Migrated = -15.21(log10 molecular weight) + 36.03

6.6 = -15.21(log10 molecular weight) + 36.03

∴ Molecular weight = 86.0 kDa

Sample B:

Distance migrated = 6.4 cm,

Distance Migrated = -15.21(log10 molecular weight) + 36.03

6.4 = -15.21(log10 molecular weight) + 36.03

∴ Molecular weight = 88.7 kDa

Sample C:

Distance migrated = 6.0 cm,

Distance Migrated = -15.21(log10 molecular weight) + 36.03

6.0 = -15.21(log10 molecular weight) + 36.03

∴ Molecular weight = 94.2 kDa

Part 2: Ion Exchange Chromatography

Part A: DEAE Column

Table 2: Absorbance value for test tube 1 to 10 and (1/10) dilution sample for determination of Hb from DEAE column

Test Tube Absorbance @ 578 nm

1 0.123

2 0.115

3 0.110

4 0.121

5 0.058

6 0.013

7 0.005

8 0.002

9 0.0012

10 0.007

1/10 dilution 0.234

Calculations: