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LAB NOTEBOOK
Running a protein gel
Context
Materials
Predictions
Protocol
Results
Reflection
7
Summary

7. Summary
It is important to summarize your methodology and observations after you have
completed an experiment. Please view a recap of this simulation and takeaway
messages regarding Running a protein gel below.

Simulation recap

 In this simulation you treated your cell culture samples with SDS sample buffer and
heated them to ensure cell lysis and the denaturation of all cellular proteins. SDS
coats the denatured proteins in negative charge.
 After loading your samples and controls on a protein gel, you separate proteins
according to molecular weight by applying an electrical current. Since the proteins
are invisible, look for the blue dye front and protein ladder reaching the bottom of the
gel as indicators of when to stop the electrophoresis.
 In order to visualize your protein bands, SDS-PAGE will typically be followed by a
Coomassie staining or a Western blot step. Western blotting ensures the specific
detection of your protein of interest, whereas Coomassie stains all proteins.

Takeaway messages
Avoid heating your samples with the lids of the tubes open since the sample buffer
would boil away, leaving your samples unusable.

Always ensure you add enough running buffer in both chambers of the
electrophoresis box to reach the wells as well as the bottom of the gel. This ensures
that an electrical current will flow to separate the negatively charged proteins by
mass.

Remove the comb from the gel wells.

You can check whether electrophoresis has begun by looking for bubbles at the
bottom of the electrophoresis box.

If you run the blue front off the gel you might lose your protein of interest.

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LAB NOTEBOOK
Running a protein gel
1
Context

1. Context
Protein gel electrophoresis is a laboratory technique that separates proteins by
mass. This technique is similar to running an agarose gel to separate and visualize
different molecules of DNA. In contrast to DNA, protein samples are first boiled in a
sample buffer containing SDS, which unfolds polypeptides and covers them with
negative charges. After boiling, the samples are loaded on a polyacrylamide gel. An
applied electrical current lets smaller proteins migrate further down the sieve-like gel
and separates them from larger ones.

After electrophoresis, the otherwise invisible proteins are typically visualized by

staining the gel with Coomassie, which generates the typical protein gel pattern of
blue bands. However, when running a sample with many proteins, such as an entire
cellular extract, the visualization of individual proteins can be very tricky as hundreds
or thousands of other proteins may obscure the protein band you are interested in.

In this case, Western blotting is employed, a common technique which allows for the
specific detection of just one protein among many with the help of a specific
antibody. Western blotting is a routine technique to test the protein expression of
specific genes in the cell.

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Materials
Predictions
Protocol
Results
Reflection
Summary