登录

需要订阅 JoVE 才能查看此.

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Protein Analysis: A Technique to Separate and Visualize Proteins Based on Molecular Weight

-- views • 1:54 min

成績單

Begin by treating the protein sample with a denaturing buffer comprising a detergent - sodium dodecyl sulfate, SDS, and a reducing agent - beta-mercaptoethanol. Heat it briefly. During this step, beta-mercaptoethanol disrupts the disulfide linkages, rendering an unfolded conformation to the protein.

The unfolded proteins with their exposed hydrophobic amino acids bind to the negatively charged SDS and acquire a uniform negative charge. Add a suitable tracking dye to the sample for real-time visualization.

Now, load the sample and standard molecular size marker in separate wells present in the stacking gel - the top layer of the polyacrylamide gel containing a lower concentration of cross-linked acrylamide polymers.

Connect the buffer-filled gel apparatus to the power supply.

In the presence of an electric field, negatively charged proteins migrate freely towards the anode and stack together to enter the resolving gel - the lower layer containing a higher concentration gel.

In the resolving gel, proteins start migrating as per their molecular size, with smaller proteins migrating rapidly, resulting in optimally resolved protein bands. Thereafter, stop the run and remove the gel.

Stain the gel with an anionic dye, Coomassie blue, that binds electrostatically to the basic amino acids in proteins, imparting them blue coloration. Thereafter, destain the gel in acetic acid solution for better visualization.

Remove the gel and identify the separated proteins based on their estimated molecular weights.

article

01:18

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis for Protein Analysis: A Technique to Separate and Visualize Proteins Based on Molecular Weight

相关视频

523 Views

JoVE Logo

政策

使用条款

隐私

科研

教育

关于 JoVE

版权所属 © 2025 MyJoVE 公司版权所有,本公司不涉及任何医疗业务和医疗服务。