polyacrylamide gel electrophoresis

Importance of Polyacrylamide Gel Electrophoresis

To prepare a PolyAcrylamide Gel Electrophoresis (PAGE) set up.

Polyacrylamide Gel Electrophoresis Method

SDS-PAGE (SDS-polyacrylamide gel electrophoresis)

Protein Electrophoresis: Theoretical Background to Know

SDS-PAGE (SDS-polyacrylamide gel electrophoresis) is an electrophoretic technique that separates polypeptide chains according to their molecular weights (Mr). The technique utilizes polyacrylamide gel containing sodium dodecyl sulfate (SDS). SDS in the sample and the gel cancels the effect of intrinsic electrical charge of the sample proteins. All proteins acquire a negatively charged rod like structure, so separation becomes largely dependent on Molecular weight of sample proteins.

Applications of protein gel electrophoresis: Once proteins have been separated by gel electrophoresis, they can be utilized for a number of downstream applications including:

1. Determine size and  isoelectric point of separated proteins.
2. Enzyme assays.
3. Further purification.
4. Immunoblotting or western blotting.
5. Elution and digestion for mass spectrometric analysis.
6. Serum protein electrophoresis is tested when a patient has an abnormal total protein or albumin blood test, or, if a patient has symptoms of diseases associated with abnormal protein production, such as multiple myeloma or multiple sclerosis.

Polyacrylamide Gel Electrophoresis Principle of Work (Protein Electrophoresis Principle)

The gel used in (SDS-polyacrylamide gel electrophoresis) can be divided into stacking gel and separating gel. Stacking gel (acrylamide 5%) is poured on top of the separating gel (after solidification) and a gel comb is inserted in the stacking gel. The acrylamide percentage is chosen in accordance with the size of target proteins in the sample. Samples are applied in wells created by the comb. Upon electric current application, proteins migrate according to their Mr, and thus are separated.

General Guidelines for Polyacrylamide Gel Electrophoresis Experiment

1. Always wear gloves to decrease protein contamination. 
2. Clean all equipment that has water and gel contact 
3. You should have a manufacturer's safety data sheet (MSDS) and precautions for all the chemicals in the lab such as Acrylamide monomer (considered as a suspected carcinogen and a neurotoxin). The safety sheets should be reviewed prior to starting the steps in the manual.