Determination of Molecular Weight via SDS Page Gel. The focus of this lab is the determination of unknown proteins via molecular weight.

Authors Avatar by standardprocess (student)

Olivia Takahara

09/10/2012

Chemistry 4046L

Determination of Molecular Weight via SDS Page Gel

Abstract

Several gel separation techniques capitalize on differences in relative protein size mobility within an applied electric field.  An unknown protein was mixed with sodium dodecyl sulfate (SDS) buffer, heated at 80°C, and loaded with a mixture of standard protein markers into a “pre-cast” agarose gel and “electrophoresed.  The gel was developed and stained. The identity of the unknown proteins were determined by comparison to the known molecular weights of the standard protein markers.

Introduction

The focus of this lab is the determination of unknown proteins via molecular weight. Several unknown proteins were run in parallel with a standard ladder. Band locations between the unknowns and the ladder were compared after staining. By comparing molecular weights of the unknowns, identities were determined.

Electrophoresis can take advantage of charged molecules and electric fields to separate proteins. Proteins are placed into a buffer constructed to induce negative charges to the proteins, which are loaded into a gel made of polyacrylamide. The gel provides a matrix of cross-linked acrylamide, which will separate the proteins as they travel through the matrix based on size/molecular weight, with molecules of smaller size traveling farther, faster. The concentration of acrylamide used to make the gel will determine the scope of molecule sizes that will be separated. A higher percentage acrylamide in a gel results in smaller pores, which will allow very small molecular weight proteins through. A low percentage of acrylamide will produce large pores and allows for more movement of larger proteins through the matrix.  Once in the appropriate apparatus, an electric field is applied to the gel. This field moves the charged proteins and they will be separated based on size, amount of charge, pore size of the gel matrix, duration of exposure to current, and strength of the electrical field.

Join now!

This experiment used the SDS-PAGE gel technique to separate proteins of interest. The presence of SDS serves to unfold proteins and allow the samples to carry charge.

 

Materials and Methods

1. Electrophoresis buffer from packet:

a. 500mL of Tris at 100mM

b .3mM SDS of pH 8 ± 0.5

c. 500 mL H2O

2. Gel, Tris HEPES SDS

a. 8-16% Precices Protein Lowell gel

3. SDS sample loading buffer (6x Reducing)

a. 375 mM, pH 6.8

b. SDS 6%

c. Glycerol 48%

d. Bromophenol 0.03%

e. MeSH 9%

4. 50/10 fixing solution 500 mL

...

This is a preview of the whole essay